Pai’s Textbook of Intrauterine Insemination Rishma Dhillon Pai, Nandita P Palshetkar, Hrishikesh D Pai
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_FM1Pai's Textbook of Intrauterine Insemination_FM2
_FM3Pai's Textbook of Intrauterine Insemination
EditorRishma DhillonPaiMD DNB FCPS DGO FICOGConsultant Gynecologist Jaslok and Lilavati HospitalMumbai, Maharashtra, IndiaCo-editorsNandita PPalshetkarMD FCPS FICOGConsultant Gynecologist and Infertility Specialist Harkisandas N Hospital, Lilavati Hospital IVF Center Medical Director, Bloom IVF EnterprisesMumbai, Maharashtra, IndiaHrishikesh DPaiMD FCPS FICOG MSc (USA)Gynecologist and Infertility Specialist Lilavati Hospital IVF Center,Mumbai, Maharashtra, IndiaFortis La Femme Hospital,New Delhi, IndiaFortis Mohali Hospital,Chandigarh, IndiaFortis Hiranandani Hospital,Vashi Navi Mumbai, Maharashtra, IndiaDY Patil Hospital,Navi Mumbai, Maharashtra, IndiaMedical Director, Bloom IVF EnterprisesMumbai, Maharashtra, India
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Pai's Textbook of Intrauterine Insemination
© 2011, Jaypee Brothers Medical Publishers
All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the editor and the publisher.
First Edition: 2011
9789350252345
Typeset at JPBMP typesetting unit
Printed at_FM5
Dr Rishma Dhillon Pai graduated from one of India's most prestigious medical institutes, The GS Medical College and the KEM Hospital, Mumbai, Maharashtra, India. She is currently an Honorary Consultant Gynecologist at the Jaslok and Lilavati Hospitals, two of the best known and reputed hospitals in Mumbai, Maharashtra, India; the Director of the Everywoman Cliniqs, Mumbai and Pearl Centre Hospital, Mumbai, Maharashtra, India which was the first to break new ground in the field of family planning.
Her special interests are infertility, minimal access surgery, contraception and adolescent problems. She trained in endoscopy, IVF and ICSI at Antwerp, Belgium. She has also trained in egg and embryo freezing at the world's busiest IVF center in Japan and also advanced her skills in this field in Italy, China and Portugal.
She has been working actively with the Obstetric and Gynaecological Societies and was elected as the Senior Vice President of Federation of Obstetrics and Gynaecology of India (FOGSI) in 2010. Notwithstanding her activity filled agenda, she is an active member of the managing committee of the Mumbai Obstetric and Gynaecological Society (MOGS), and Indian Association of Gynaecological Endoscopists (IAGE). She is the treasurer of the Indian Society for Assisted Reproduction (ISAR). She was also the Chairperson of the Food, Drugs and Medicosurgical Equipment Committee of FOGSI (2004–2008). She has organized 14 conferences as organizing Chairperson or Secretary.
She was among the first doctors in India to start the procedure of uterine balloon ablation therapy for _FM6heavy bleeding. She is one of the first in Asia to offer a nonsurgical treatment for the common problem of fibroids, using MRI-guided focussed ultrasound. She was one of the first in India to start and teach other gynecologists—the use of abortion pills. She also holds the distinction of having delivered a 60-year-old woman, one of the oldest patients in India.
She has been an invited faculty at more than 180 national and international conferences. She is also actively involved in training postgraduate students. She has to her credit several academic contributions, having contributed chapters to over 38 books and published over 33 articles in leading journals and newsletters. Her career, which has spanned nearly two decades, has brought her numerous accolades and firmly established her among India's most prominent and popular obstetricians and gynecologists. In 2011, she was awarded the Newsmakers Achievers Award for best gynecologist. In 2010, she has been the recipient of the GR8 Women Achievers Award for excellence in the field of medicine; in 2009, she was awarded the Navshakti Awards for excellence in the field of medicine; in 2008, she received the award for Distinguished Alumni, awarded by Jaihind College, Mumbai, Maharashtra, India; she has won many prizes for papers and presentations in the field of gynecology.
She works untiringly towards women's health issues, balancing the demands of her busy practice with her voluntary and advisory capacities. She leads the team of wonderful women who follow their dreams, work hard and try to achieve the peak of perfection in their area of expertise._FM7
Dr Nandita P Palshetkar is a Professor in Obstetrics and Gynecology, DY Patil Medical College, Navi Mumbai, Maharashtra, India and also is a teacher for superspecialty degree (FNB), Fellowship in Reproductive Medicine, National Board, Delhi, India, at Lilavati Hospital, Mumbai, Maharashtra, India. She has trained in IVF and Micromanipulation from the University of Ghent, Belgium; Alpha School of Embryology, Naples, Italy; IVF and ICSI Training, Melbourne, Australia; Copenhagen, Denmark (IVF Centers).
She has an endless list of affiliations, the most significant ones being: Senior Vice President, FOGSI (2011); Chairperson, Perinatology Committee, FOGSI (2004–2008); Librarian, Mumbai OB/GYN Society; Secretary Association of Maharashtra OB/GYN Societies; Managing Committee Member, Indian Association of Gynecological Endoscopy; Managing Committee Member, Indian Society of Assisted Reproduction; Member, National Guidelines for Accreditation; Supervision and Regulation of ART Clinics in India (2006–2007).
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Dr Hrishikesh D Pai amongst the initial pioneers in the field of infertility in India. He has been the first doctor in India to have introduced several new techniques in this discipline. He balances his demanding private practice with various associations as being the present Vice President of the Indian Society of Assisted Reproduction; Treasurer of Federation of OB/GYN Societies of India; The Adjunct Assistant Professor in Clinical Embryology and Andrology at the Eastern Virginia Medical School, Norfolk, USA.
In the past, he has held prestigious posts as senior Vice President of Federation of OB/GYN Societies of India (FOGSI—2006); President of the Mumbai Gynecological Society (MOGS—2008); President of the Indian Association of Gynecological Endoscopists (2007–2009); Postgraduate Teacher in Gynecology at the DNB examinations, National Board, New Delhi, India; Assistant Honorary Consultant, K Bhabha Municipal General Hospital (1991–2007); and Assistant Honorary Professor, Dr DY Patil Medical College, Mumbai University (1993–2007), Mumbai, Maharashtra, India.
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_FM11Contributors_FM13Preface
It is indeed a great pleasure to write the preface for Pai's Textbook of Intrauterine Insemination.
Intrauterine Insemination (IUI) is one of the safest, least invasive and most cost-effective forms of assisted reproduction. The procedure is simple and very easy to perform. There are several indications of IUI including male factor, female factor or combined factors.
The book has been compiled so as to provide a step-by-step approach in dealing with infertile patients requiring IUI treatment, starting with patient selection, counseling, ovulation induction protocols, various sperm washing techniques and clinical aspects of IUI. The book also contains chapters detailing all relevant information about setting up of an IUI lab and legal and ethical aspects related to donor insemination.
We feel that the simple and systematic approach provided in this book and its easy reading style will help to make it one of the popular manuscripts amongst the general gynecologists starting this technique or trying to achieve perfection in it.
Rishma Dhillon Pai
Nandita P Palshetkar
Hrishikesh D Pai

Introduction to IUI1

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INTRODUCTION TO IUI
IUI is considered as the first line of treatment when applicable, in many infertility centers as it is a simple and inexpensive procedure. Intrauterine insemination (IUI) has a long history 1, 2, 3, 4. It was introduced nearly 200 years ago by John Hunter in the 1770's. Since then it has undergone many changes and advances till date.
In Intrauterine insemination (IUI) washed and prepared sperms are introduced into the woman's uterus, timed with ovulation, with the aim of getting the sperms nearer to the ovum.
It is often used as the first line of treatment for couples with “unexplained infertility”. It is also recommended for women with mild endometriosis. IUI can also help couples who are not able to have intercourse because of difficulties such as premature ejaculation or disability, as sperm is introduced directly inside the uterus. Women whose husbands have total testicular failure, have no option but to use donor insemination in order to achieve pregnancy. IUI with donor sperm is being increasingly used by women who intend to become single mothers.
In the past, IUI was done without ovarian stimulation by fertility drugs (“unstimulated cycle” or “natural cycle” IUI). In unstimulated cycles, IUI is timed with natural ovulation, which is usually detected by monitoring of urinary LH, using LH kits, starting daily from day 8 of the periods. IUI is done 12–16 hours after the LH surge is detected by such LH kits. Nowadays, IUI is often combined with fertility drugs (“the stimulated cycle”). Ovulation induction is often recommended in anovulatory 3infertility like PCOD or unexplained infertility and also in mild male factor and mild endometriosis cases. These drugs increase the chances of pregnancy by multifollicular development and by producing good quality oocytes. The drugs commonly used are clomiphene citrate, letrozole and injectable gonadotropins. Usually these medicines are given starting from day 3–5 of the menstrual cycle. Concurrently serial transvaginal sonography is done to monitor follicular development from day 6–8 of the period. When the leading follicule becomes 18–20 mm in size, hCG (human chorionic gonadotropin) trigger is given. IUI is planned 36–40 hours after the trigger. The husband is asked to collect a semen sample, preferably in the semen collection room in the vicinity of IUI lab, or alternatively, he can get the sample in a clean sterile, non toxic container from home. The sample should reach the laboratory within 30–45 min. The best quality and most motile sperms are harvested by one of the different semen preparation techniques. The washed sperms are deposited in the uterine cavity by a flexible catheter. The patient is made to rest for about 15 minutes after the procedure. A pregnancy test (preferably Serum β-hCG) is done 14 days after the IUI.
The success rate of IUI varies from 5–25% depending upon the age of the female partner, the extent of male factor, the cause of infertility and the ranking of the laboratory which does the semen preparation (as assessed by the equipment, maintainance, and staff skills). IUI can help couples with low sperm count or poor motility. A post wash count of more than 5 million per ml and 50% progessive motility usually leads to good success rates.
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The success rates for IUI with natural cycle is 5–7%, and with fertility drugs it is about 15 % per cycle. Of course, as with any form of infertility treatment, there are variable factors which can affect how successful this treatment will be. It is not applicable for couples with:
  • Tubal blockage or severe tubal damage
  • Ovarian failure
  • Advanced stages of endometriosis
  • Severe male factor infertility
The disadvantages of ovarian stimulation for an IUI cycle are the associated risks of ovarian hyperstimulation syndrome (OHSS) and multiple pregnancies.
But overall, it is a reasonable initial treatment that should be utilized for a maximum of about 3–4 cycles, where applicable, it being relatively simple, inexpensive and effective.
REFERENCES
  1. CohenMR. Intrauterine insemination. Int J Fertil 1962; 235.
  1. WhiteRM, GlassRH. Intrauterine insemination with husband's semen. Obst Gynaecol 1976: 47: 119–23.
  1. BarwinBN. Intrauterine insemination using husband's semen. J Reprod Fertil 1974; 36: 101–3.
  1. HansonFM, RockJ. Artificial insemination with husband's sperm. Fertil Steril 1951 2:162–4.

Indications, Patient Selection and Work-up Before IUI2

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Appropriate patient selection is the most important factor which determines the success of any treatment. With IUI, many a times the treatment is empirical, still it is possible to select a group of couples where the treatment will be significantly beneficial. The rationale behind the treatment is to deposit a good number of highly motile and morphologically normal sperms in the uterus near the fundus at the anticipated time of ovulation, bypassing factors which depend upon deposition of sperms in vagina and transport through the cervical mucus to the upper genital tract. There are several indications for IUI which may be male factor infertility, female factor or combined factors.
A complete work-up is required before taking up the patient for IUI. Any contraindications to the procedure must be ruled out. Patients should be counseled about the procedure involved, success rates, other options and risks associated.
 
INDICATIONS OF IUI
 
Male Factors
 
Impotence/ejaculatory dysfunction
This can be due to a number of causes.
  • Anatomical: Hypospadias— Here deposition of semen occurs outside vagina or much away from the os. In such patients semen is collected by masturbation for IUI.
  • Neurological: This can be due to:
    • Spinal cord injury
    • Diabetes mellitus
    • Multiple sclerosis
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    • Atherosclerosis
    • Damaged hypogastric nerves during surgeries like abdominoperineal resection of rectum, retroperitoneal lymph node dissection and aortoiliac surgery.
In these conditions, the sperm quality especially its motility is hampered despite high sperm density. Furthermore debris, inflammatory cells and quite often bacteria abound in these samples. The success of treatment depends upon sperm quality. Good results are obtained with samples where the progressive motility is more than 20–30%.
 
Retrograde Ejaculation
In this condition, there is reflux of semen backwards from the posterior urethral valve into the bladder, at the time of ejaculation. The sperms lose their viability due to toxic effects and acidity of urine. It can be due to diabetes mellitus, multiple sclerosis, drugs like α adrenergic blockers and phenothiazines and damage to innervation of bladder neck during surgeries like TURP (Transurethral resection of prostate), and retropubic prostatectomy. In retrograde ejaculation, urine is centrifuged and then washed to isolate sperms and then IUI is performed.1
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Psychological conditions: Such patients need sex-psychotherapy. Drugs such as viagra or papaverine may be given to bring about a good erection. Some patients benefit with the use of mechanical vibrators. Very occasionally the patients may have to be subjected to general anesthesia and electroejaculation.
Drug induced: Drugs like sedatives, antidepressants, antihypertensive agents, cimetidine etc. can cause ejaculatory dysfunction.
 
Subnormal Semen Parameters
This includes:
  • Oligozoospermia
  • Asthenozoospermia
  • Teratozoospermia
  • Hypospermia
  • Highly viscous semen.
The cause of infertility in such conditions is decreased availability of normal motile sperms for fertilization. As defined by WHO, a normal semen sample has a sperm count of more than 20 million/ml, with 50% or more of them showing forward progression, and 30% or more having normal morphology.2
Ideally a total motile pre-wash count of more than 10 million or a post-wash motile sperm count of five million is necessary to achieve a good pregnancy rate. Additionally percentage motility of more than 40% in the final semen preparation correlates well with favorable outcome.
Patients with severe male factor infertility should go directly for Intracytoplasmic Sperm Injection (ICSI) or the use of donor sperms for insemination (AID).
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Other Factors
Though the main treatment for obstructive azoospermia is percutaneous epidydimal sperm aspiration (PESA) with ICSI, there is a recent report of achieving pregnancy after extracting sperms with PESA and performing IUI. Other conditions such as allergy to semen, vaginismus and other sexual dysfunctions may be treated with IUI.
HIV infection: Sperm washing can significantly reduce the viral load.3 Recently insemination of HIV negative women with processed semen sample of HIV positive partner has been carried out to reduce the risk of transfer. However, prepared semen sample should be tested by PCR before insemination.
 
Female Factors
 
Ovulatory Dysfunction
It contributes to 30–40% of the female factor. In these cases the first choice would be ovulation induction combined with timed intercourse or IUI. Many studies have shown that IUI gives better results as compared to timed intercourse.
 
Cervical Factor
The cervix plays an important role in achieving successful pregnancy. It performs the following functions:
  • Control of sperm entry into the upper genital tract
  • Protection of sperms from vaginal acidity
  • Nutrition of sperms
  • Selection of sperms based on motility
  • Sperm reservoir function
  • Initiation of capacitation
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The following are some common causes of cervical factor infertility:
  • Insufficient mucus production, which may be due to previous cauterization, surgery or rarely cystic fibrosis.
  • Altered quality of mucous
  • Abnormal cervix: Stenosis, injury, malformation, infection, erosion
  • Abnormal postcoital test (PCT) or hostile cervical mucous (the general consensus is that PCT has non-predictive value in terms of pregnancy)
IUI helps bypass these hostile factors. It has been observed that only 0.1% of the sperms placed in vagina are present in the cervical canal one hour after insemination.4 Direct deposition of motile sperms in the uterine cavity can reverse this situation, and increase the chance of pregnancy. The use of IUI in patients with cervical factor infertility, yields very good pregnancy rates with the success rate of 14 to 18%.
 
Endometriosis
IUI with ovulation induction can be tried in cases of mild endometriosis. Patients with mild to moderate endometriosis have good pregnancy rates between 7–18%. However, as the pregnancy rates (3–5%) are very low with severe endometriosis, it is best to opt for IVF/ICSI.
 
Common Factors
 
Immunological
Antisperm antibodies can be found in both males and females. Causes in men are usually testicular trauma or 11obstruction to the male genital tract. In women, it can happen due to a break in the vaginal epithelium, peritoneal instillation, anal or oral intercourse. These antibodies prevent binding of sperm to zona pellucida and also impair the sperm movement. Various treatments like prolonged use of condoms, immunosuppression with steroids and laboratory procedures to wash sperm have been tried. However all these have limited success.
Both IUI and IVF have shown to have high pregnancy rates in such patients. IUI helps to bypass these antibodies in cervical mucus.
 
Unexplained Infertility
This diagnosis is made when a couple fails to conceive despite there being no obvious cause, even after subjecting the patient to a complete work-up. The diagnostic protocol should include an assessment of ovulation, evaluation of tubal patency and a normal semen analysis. The average incidence of unexplained infertility is around 10–15%.
Defects in folliculogenesis, gamete development, fertilization, and embryo development may be the factors responsible. The rationale of empirical therapy is to bypass these causative factors. The managing principles are:
  • Increasing availability of gametes by ovulation induction
  • Improving gamete quality
  • Bringing the gametes together by IUI or IVF.
The efficacy of various treatments in unexplained infertility is5:
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Treatment
Combined pregnancy rate per initiated cycle
No treatment
1.3%
IUI
3.8%
CC
5.6%
CC with IUI
8.3%
HMG
7.7%
HMG with IUI
17.1%
IVF
20.7%
 
Insemination with Husband's Frozen Semen
This is required in the following conditions:
  • Absentee husband
  • Antineoplastic treatment
  • Vasectomy
  • Poor semen parameters
  • Drug therapy
 
Insemination with Donor Sperms
It is now mandatory to use cryopreserved donor samples in order to minimize risk of HIV transmission. The indications for insemination with donor semen are:
  • Azoospermia
  • Severely subnormal semen parameters
  • Hereditary disease in father
  • Persistent IVF/ICSI failures
  • Rhesus isoimmunization
  • Patient unable to afford ICSI.
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Patient Selection and Work-up
An appropriate patient selection is the key to success for any treatment. A complete work-up including a detailed history is required before taking a patient for IUI. Many infertile couples have more than one contributory factor which should be identified at the earliest. A scientific approach is warranted for a complete and efficient evaluation of female and male factors. More importantly, any contraindications to the procedure should be ruled out, as these can compromise the results. Apart from a detailed history and physical examination, and routine investigations certain specific tests for both the partners are required.
 
EVALUATION OF THE FEMALE PARTNER
 
Routine Investigations
CBC, ESR, sexually transmitted disease (VDRL, HBsAg, HIV) blood sugars, urine routine, bleeding and clotting time.
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Anthropometric measurements such as BMI (Body mass index) and WHR (Waist to hip ratio) will help to identify patients with central adiposity. These patients may require further evaluation of hyperandrogenism and hyperinsulinemia that may cause aberration in ovulation and cause luteal phase deficiency despite medication.
 
Hormonal Investigations
 
Serum FSH, LH, estradiol on day 2/3 of cycle
  • FSH > 10 mIU/ml and E2 > 60 pg/ml indicates poor ovarian reserve
  • LH/FSH > 2/1 indicates PCOS
  • Low LH, FSH, E2 indicates hypogonadotrophic hypo­gonadism
  • FSH more than 10 mIU/ml on day 10 after giving a clomiphene challenge indicates poor prognosis
In case of patients suspected to be poor responders, one can do these additional tests:
  1. Serum inhibin – B test which is > 45 pg/ml in poor responders.
  2. Clomiphene citrate challenge test:
    CC 100 mg per day from day 5 to day 9 and FSH on day 10. A high FSH (>10 mIU/ml) indicates poor response and poor prognosis. This also points towards direct stimulation with gonadotropins instead of clomiphene citrate.
  3. Serum AMH (antimullerian hormone)
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Serum Prolactin and T3 /T4/ TSH
In case of patients with PCOS diagnosed by USG/symptomatology/clinical or biochemical features of hyperandrogenism, the following tests are indicated:
  1. Fasting serum insulin level (>10 mIU/ml is significant)
  2. Fasting and Postprandial blood sugars.
  3. Fasting blood glucose/fasting insulin ratio (>4.5 is normal)
  4. DHEAS, androstenedione and testosterone
  5. In obese patients, a follicular phase 17-OHP (17-Hydroxyprogesterone) level (to rule out congenital adrenal hyperplasia) and dexamethasone suppression test (to rule out Cushing's syndrome) should be carried out.
  6. Rarely serum alanine transaminase level is done in patients who are intolerant to metformin treatment and who need to be placed on rosiglitazone.
  7. In women with past history of renal disease on metformin treatment, serum creatinine and/or 24 hr creatinine clearance may have to be done.
For screening and academic purposes a C-peptide assay may be performed to pick up latent diabetes.
 
Tests for Ovulation (Ovulatory or Anovulatory)
  • Basal body temperature
  • Serial vaginal ultrasound follicular scan in a spontaneous cycle
  • Serum progesterone on day 21 of cycle >4 ng/ml indicates ovulation and >10 ng/ml indicates adequate luteal phase.
  • Urinary LH surge testing.
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Pelvic Sonography
This helps in evaluating uterus, uterine cavity and adnexae. Ovarian volume, antral follicle count and presence or absence of PCO pattern should be noted.
HSG: This is done just after the means and over. It helps in evaluation of uterine cavity and to check the tubal patency.
Diagnostic laparoscopy and hysteroscopy may be required in certain cases to establish the exact diagnosis (especially endometriosis).
Physical parameters
Clinical parameters
Endocrinological
Weight (kg)
USG – evaluation of uterus and cavity
Day 2/3 FSH, LH and E2
Height (Meters)
Measurement of ovarian reserve
S. Prolactin
Body mass index (kg/m2)
Number of antral follicles
Fasting Insulin, SHBG and DHEAS
Waist to hip ratio
Diagnostic laparoscopy and hysteroscopy for evaluation of cervical, tubal, uterine and ovarian factors
Day 21 progesterone
 
Tests to rule out Tuberculosis
These are especially important in developing countries. These include:
CBC with ESR
Chest X-ray
Mantoux test
Endometrial biopsy
TB ELISA IgG and IgM
TB-PCR
BACTEC
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EVALUATION OF MALE PARTNER
  • Detailed history and examination
  • Built
  • Hair distribution scoring
  • Gynecomastia
  • Examination of testis, vas, epididymis, volume of testis in case of azoospermia
  • Semen analysis and culture
 
PREREQUISITES FOR IUI
  • Age less than 40 years
  • Patient capable of spontaneous or induced ovulation
  • At least one patent fallopian tube with good tubo-ovarian relationship
  • Sperm count of more than 10 million/ml pre-wash or a post-wash count of > 3–5 million motile sperms with percentage motility of more than 40%
  • Easy access to the uterine cavity via a negotiable cervical canal.
 
 
Case 1
30-year-old female, married for 5 years with primary infertility. She had regular cycles and has undergone many cycles of follicular monitoring which showed dominant follicle rupture on day 15 of cycle. Her hormonal profile was normal and HSG showed a normal uterine cavity with bilateral free spillage of dye. Husband semen analysis showed mild oligospermia with normal motility. The patient underwent ovulation induction with letrozole 5 mg from day 3 to day 7 with follicular monitoring and 18intrauterine insemination. She conceived in her 2nd cycle of treatment and delivered a healthy female child at term.
 
Case 2
28-year-old female, married for 3 years with irregular menses with primary infertility. USG showed evidence of polycystic ovaries. Hormonal profile revealed no abnormality. HSG showed a normal uterine cavity with bilateral free spillage of dye. Husband's semen analysis showed normal count and motility. The patient underwent ovulation induction with clomiphene citrate 100 mg from day 2 to day 6 of her periods. Transvaginal sonography on day 8 showed a single dominant follicle. This was followed by serial transvaginal scans. The ovulation was triggered when the dominant follicle reached 18 mm followed by IUI 36 hours later. A β-hCG 14 days later was 340 mIU/ml and sonography at 6 weeks showed a single intrauterine gestation sac.
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REFERENCES
  1. ZhaoY, GarciaJ, JarowJP, et al. Successful management of infertility due to retrograde ejaculation using assisted reproductive technologies: a report of two cases. Arch Androl 2004; 50(6)391–4.
  1. World Health Organization Laboratory Manual for examination of human semen and sperm cervical mucus Interaction, 4th Cambridge University Press  1999.
  1. BujanL, PasquierC, LabeyrieE, et al. Insemination with isolated and virologically tested spermatozoa is a safe way for human immunodeficiency type 1 virus serodiscordant couples with an infected male partner to have a child. Fertil Steril 2004; 82: 857–62.
  1. SettlageDSF, MotishimaM, TredwayDR. Sperm transport from external cervical os to fallopian tube in women; a time and quantitative study. Fertil Steril 1973, 24 55–61.
  1. GuzickDS, CarsonSA, CoutifarisC, et al. Efficacy of super ovulation and intrauterine insemination in treatment of infertility. National Cooperative Reproductive Medicine Network. N Engl J Med 1999, 340:177–83.

Normal Semen Analysis and CASA3

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A semen analysis evaluates certain characteristics of a man's semen and the sperms contained in the semen. It is important not only for the diagnosis of male infertility, but is also important for deciding upon treatment for the infertile couple. It may also be done after a vasectomy to verify that the procedure was successful.
 
INSTRUCTIONS TO THE COUPLE
The couple must be instructed to abstain from intercourse or masturbation for 2 to 3 days before the test. Preferably, the collection must be done in the laboratory itself. However, as this may not be practical, collection can be done at home, into a clean, wide mouthed, container. The sample must reach the laboratory ideally within one hour of collection1.
It is very important to remember these points. Often, normal semen samples are erroneously diagnosed as abnormal, because one or more of the above precautions has been forgotten.
 
COLLECTION OF SPECIMEN
The most common way to collect a semen sample is through masturbation, directing the sample into a clean container.
A sample may also be collected during intercourse in a special type of condom known as a collection condom. Collection condoms are made from silicone or polyurethane, as latex is somewhat harmful to sperm. A third option for collecting a sample is through coitus interruptus 23(withdrawal). With this technique, the man removes his penis from his partner near the end of intercourse and ejaculates into a container. However, this should not be used routinely as the initial sperm rich portion may be lost. This technique is mostly only recommended for men testing for success of a vasectomy, in contrast to an investigation of infertility.
Finally, if a blockage in the vas deferens is suspected to impede fertility, semen can be taken directly from the epididymis. Such a collection is called percutaneous epididymal sperm aspiration (PESA). Alternatively, the testicular tissue itself, instead of the sperm produced can be investigated. Then, the collecting method is called testicular sperm extraction (TESE).
 
PHYSICAL PARAMETERS
Before examination the sample is thoroughly mixed with a pipette.
 
Coagulation
Freshly ejaculated semen sample is usually in the form of thick coagulum. This is because of the enzyme protein kinase secreted by seminal vesicles. It is absent in cases of congenital absence of vas deferens, ejaculatory ducts or seminal vesicles. A normal semen sample liquefies within 20–30 minutes at room temperature.
 
Liquefaction
Liquefaction is the natural change in consistency of semen from a semi-liquid to a liquid state. The significance of 24liquefaction is that the sperms which are contained in a gel like matrix before liquefaction get released and distributed evenly throughout the sample. Incomplete liquefaction may give false results during semen analysis.
Liquefaction is completed within 15 to 30 minutes and is because of the enzyme fibrinolysin secreted by the prostate gland. Delayed liquefaction (>60 min) is because of prostatic dysfunction.
 
Appearance
A normal semen sample has a homogenous gray opalescent appearance. It can become yellow with increased abstinence. A translucent appearance is associated with low sperm counts.
 
Odor
The musty odor of semen is due to the presence of spermin secreted from the prostatic gland.
 
Volume
Normal volume is 2 to 6 ml. Volume less than 1 ml is considered as abnormal.
Causes of low volume of semen are:
  1. Faulty collection
  2. Failed emission
  3. Short abstinence interval
  4. Obstruction of ejaculatory tract
  5. Congenital absence of the vas
  6. Retrograde ejaculation
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A post-ejaculatory urine analysis to look for retrograde ejaculation is indicated when ejaculate volume is less than 1 ml, except in cases of hypogonadism, congenital bilateral absence of vas deferens, collection problems or when short abstinence intervals are present.
 
Viscosity
Viscosity refers to the tendency of the liquefied semen sample to form discrete drops from the tip of the pipette. If drops do not form freely, the semen cannot be drawn into the pipette and the viscosity is considered to be high.
Viscosity is graded as follows:
  • Normal: If semen is released as a single drop within a distance of 2 cm from the tip of the pipette.
  • Minimally increased: If strands of 2 to 4 cm are formed.
  • Increased: Strands of 2 to 8 cm.
  • Grossly increased: Strands of >8 cm
Causes of abnormally increased viscosity are:
  1. Infection causing abnormal prostatic function.
  2. Artifact due to unsuitable plastic container.
  3. Anti-sperm antibodies.
  4. Frequent ejaculation
  5. Psychological state of patient.
In case the viscosity is high, it may be reduced by aspirating the semen sample in and out of a syringe fitted with an 18 gauge blunt needle. Treatment with an enzyme such as chymotripsin may also decrease the viscosity.
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pH
The pH should be measured within one hour of ejaculation. Normal pH is between 7.2 to 7.8. A more alkaline pH is suggestive of an acute infection, and acidic values are due to obstruction and chronic infection.
 
Normal Semen Parameters as Suggested by WHO
Parameter
Normal value
Abnormality
1. Volume
2.0 ml or more
Hypospermia/Aspermia
2. pH
7.2 to 7.8
3. Count/ Concentration
20 million per ml or more (> 40 million per ejaculate)
Oligospermia: count <20 mil Azoospermia: no sperms
4. Motility
50% or more forward progressive motility
Asthenospermia (reduced motility)
5. Morphology (by Pap staining)
30% or more normal looking sperms
Teratozoospermia
6. Viability
50% or more viable
Necrozoospermia
7. Leukocytes
< 1 million per cc
8. Fructose content
150–600 mg/100 ml
Or
>13 µmol/ml
Decrease is s/o seminal vesicle block/vas deferens absence or block
 
Initial Microscopic Examination
When the aliquot of semen is placed on the counting chamber, the count and motility should be determined immediately. This will prevent the effect of heat from the microscope light source influencing the results. A phase contrast microscope is recommended for all examinations 27of unstained fresh or washed semen. A fixed volume of 10 microliter of semen is delivered onto the slide and covered with a cover slip 22 × 22 mm. Initially it is seen under 100 X for mucus strands and spread of spermatozoa. Then the magnification is increased to 400 X. If no sperms are seen the sample should be centrifuged, the sediment is re-suspended and examined. The following are recorded:
 
Agglutination and Aggregation
  • Agglutination is caused by multivalent sperm antibody and usually involves live sperms. Only sperms adhere to each other without participation of other cells or debris.
  • Aggregation involves only dead sperms, other cells and debris and its presence is considered abnormal.
 
Other Cells and Debris
Some form of debris is usually present like:
  • Epithelial cells: May be present from the urethra
  • RBCs: Should not be normally seen
  • Bacteria and protozoa may be noted.
  • Basic sperm morphology defects like coiled tails, duplicate heads, etc should be noted.
  • Round cells: This may be leukocytes or immature sperms.
 
METHODS OF ASSESSMENT OF SPERM CONCENTRATION
Two methods are commonly used in practice:
  1. Makler chamber
  2. Hemocytometer
28
 
Makler Chamber
 
Chamber Description
The Makler counting chamber is only 10 microns deep: 1/10th of the depth of ordinary hemocytometers, making it the shallowest of known chambers. Constructed from two pieces of optically flat glass, the upper layer serves as a cover glass, with a 1 sq mm fine grid in the center subdivided into 100 squares of 0.1 × 0.1 mm each. Spacing is firmly secured by four quartz pins (Figs. 3.1A and B).
A drop of the semen sample is then placed in the Chamber and counting initiated: Sperm heads within a ten square area are counted in the same manner as blood cells are counted in a hemocytometer, their number represents their concentration in millions per ml. In cases of oligospermic semen, sperms in the entire grid area are to be counted, representing their concentration in hundreds of thousands.
The chamber is easily rinsed with water for reuse. Contact surfaces are wiped with special lens paper after washing.
 
Advantages of using Makler Chamber
  1. Applied sperms are uniformly distributed and monolayered, and are observed in one focal plane, eliminating blurring.
  2. Dilution is unnecessary even with concentrated specimens. Analysis is done directly from original specimen in its natural environment.
  3. All spermatozoa acquire free, frictionless horizontal movement and are always examined under constant standard conditions.
    29
    Figs 3.1A and B: Makler chamber method of sperm counting: A. Makler instrument B. Counting method
    30
  4. The specimen can be analyzed quickly as an office procedure while the patient is waiting, and even by an inexperienced person.
  5. Accuracy of analysis is enhanced through the elimination of the various steps required by the usual hemocytometric technique. In addition, the fact that sperm motility is examined each time under identical conditions further increases accuracy. Errors incurred by uncontrolled pressure applied to the cover slip are thus avoided.
  6. The 10-micron depth of the Makler chamber is ideal for still or movie camera photomicrography, as it approximately matches the field depth of the objective used in semen analysis.
  7. The chamber is quickly and easily available for reuse. In a busy laboratory a large number of tests per hour can be made by a single technician.
 
Hemocytometer
10 microliters of diluted specimen is transferred by touching the edge of the cover slip on Neubauer's hemocytometer. Phase contrast microscope is used at a magnification of 40 X. Only normal spermatozoa are included in the count.
  • If the sample contains less than 10 sperms per large square, all 25 squares are assessed.
  • If 10–40 sperms per large square are present, 10 large squares are assessed.
  • If sample has >40 spermatozoa per large square, 5 large squares are assessed.
31
The disadvantage of this method is that large dilutions have an effect on precision of results.
It is important to understand that the count varies widely, even among normal fertile men. Hence, a man cannot be considered oligospermic till multiple samples have been evaluated. Variations may be due to biological or technical reasons. The initial diagnosis of azoospermia is established when no spermatozoa can be detected on high powered microscopic examination of a pellet after centrifugation of the seminal fluid on at least 2 separate occasions.3 The World Health Organization recommends that seminal fluid be centrifuged for 15 minutes at 3000 g or greater.2
 
SPERM MOTILITY
 
Techniques for Evaluation of Sperm Motility
There are several means for evaluating motility. Which technique to use depends on experience of the operator, the desire for precision and repeatability, and availability of equipment. In almost all cases, motility is reported as the percentage of sperm that manifest motility (usually progressive motility). The probability of conception increases with increasing motility up to 60%.4
 
Manual Motility Estimates (Wet Mount)
This commonly-used technique involves placing a sample of diluted semen on a microscope slide, examining it with a microscope and estimating the fraction of the population that is motile.
32
More specifically, a sample of semen is diluted in warm extender or buffered saline, and about 10 to 20 microliters of this sample is pipetted onto a clean, prewarmed microscope slide. A cover slip is lowered onto the sample, avoiding formation of air bubbles if possible, and the slide is examined using a microscope with a 20X objective. At least ten widely-spaced fields are examined to provide an estimate of the percentage of motile sperms.
A bright field microscope can be used for evaluating motility if the field diaphragm is closed to enhance contrast and ability to visualize sperm. A much better choice is a phase contrast microscope or a microscope equipped for differential interference contrast (DIC).
Manual motility estimates are easy to perform and require minimal equipment. In the hands of an experienced evaluator, manual estimates generally provide good estimates of motility. The chief limitation of this technique is its subjective nature.
Sperm motility is graded by visualizing at least 100 spermatozoa in different fields, as follows:
  • Rapid linear (forward) motility: Grade A
  • Slow forward motility: Grade B
  • Nonprogressive motility: Grade C
  • No motility: Grade D
Normal motility, at the end of 1 hour after ejaculation, is defined as:
  1. 50 percent or more motile (Grade A+B) or
  2. 25 percent or more Grade A
33
 
SPERM MORPHOLOGY
In keeping with the move to a more strict definition of sperm morphology, WHO has redefined what they consider to be a normal spermatozoon and subsequently set an empirical reference value of 30% normal forms and above as “ normal”.
Staining of the sample is usually done by Papanicolaou stains. A normal spermatozoon has an oval head shape with regular outline and a well-defined acrosomal region covering 40–70% of head; vacuoles occupy less than 20% of the head area (Fig. 3.2).
Fig. 3.2: Normal sperm morphology
34
The head: length: 4.5–5 µm, width: 3.0–3.5 µm and length/width ratio: 1.5–1.75; no cytoplasmic droplets more than 1/3 of the size of a normal sperm head.
No dimensions and no description of a normal midpiece are mentioned. Defects are described, e.g. insertion of the tail in more than 90% of the head's longitudinal axis is abnormal. No dimensions and no description of a normal tail are mentioned, only defects are described (Fig. 3.3).
Another important evaluation aiming to clarify male fertility is the strict criteria for sperm morphology (Kruger's or Tygerberg's criteria 1986)5.
Strict criteria of sperm morphology established by Kruger et al define normal spermatozoa as having an oval configuration with a smooth contour.
The head: length is 4.5–5 µm, width: 3.0–3.5 µm and length/width ratio: 1.5–1.75. The acrosome is well-defined, comprising 40–70% of the distal part of the head. No abnormalities of the neck, midpiece or tail and no cytoplasmic droplets of more than half of the sperm head are accepted. Borderline forms are considered abnormal.
All other abnormal sperm forms—round, small, large, tapered, double head, double or coiled tail, cytoplasmic droplets—are classified following the WHO classification.
Normal and borderline forms grouped together are called ‘the morphology index’. Patients with a morphology index less than 30% will have a severe reduction in fertilization as compared with patients having an index greater than 30%. In Kruger's practice, the normal forms considered alone are called the ‘percentage of ideal forms’ (PIF). A PIF greater than 4% is considered favorable and less than 4% unfavorable.
35
Fig. 3.3: Abnormal sperm morphology
At least 200 cells per slide are to be evaluated. A micrometer in the eyepiece of the microscope is used for routine measurements.
Sperm viability: It is assessed by using supravital staining techniques. Dead cells with damaged cell membranes take up the stain, whereas live cells remain unstained.
36
Some other parameters which are not included in the routine semen analysis report are:
  1. Tests for antisperm antibodies: These tests look for the presence of agglutination in the semen sample. Commonly done tests are the Immuno bead test (IBT) and the mixed agglutination reaction (MAR).
  2. Acid phosphatase level: Normal range 25,000 to 60,000 IU/ml.
  3. Zinc: Normal range 90 to 250 mg/ml.
 
FRUCTOSE LEVEL
The level of fructose in the semen is measured. The normal level is at least 3 mg/ml. WHO specify a normal level of 13 μmol per sample. Absence of fructose may indicate a problem with the seminal vesicles, or an obstructive pathology.
 
RECENT ADVANCES IN SEMEN ANALYSIS
 
CASA
Computer aided semen analysis (CASA) eliminates the subjective error in examination of samples which is of great benefit. Also, results are available much faster, and more samples can be processed in a short time.6
Computer-assisted techniques are most often used for the assessment of sperm concentration and mobility characteristics, such as velocity and linear velocity. Although many systems can give very accurate information about motility patterns of motile sperm, immotile sperm cannot accurately be distinguished from other cells, particles or 37debris without addition of staining. Now-a-days, there are CASA systems, based on image analysis and using new techniques, with near perfect results, and doing full analysis in a few seconds. In a prospective study, the sensitivity of CASA for prediction of fertilization was high (74%), whereas the specificity was low (40%). Therefore, CASA of washed spermatozoa may help in identifying couples who would benefit more from ICSI than IVF.7
 
CASA Systems
SQA-V: The SQA-V, also known as the ‘Sperm Quality analyzer or spermalite, is a high performance sperm analysis instrument used to test male fertility. It combines electrooptics, computer algorithms and video microscopy to provide a precise and accurate 75 second automated semen analysis.
ISAS: Integrated semen analysis system is a CASA system based on image analysis, also known as Proiser.
IVOS sperm analyzer: The integrated visual optical system (IVOS) was developed by Hamilton Thorne, the leading manufacturer of CASA systems since 1986. The IVOS is unique in that it is the only CASA system that integrates the optical system within the unit, so that an external microscope is not needed. Samples are placed on a computer controlled, heated stage which maintains samples at the proper analysis temperature and automatically moves to selected fields for analysis.
However, persistent questions about the validity and reproducibility of results have kept CASA from becoming standard procedure in the andrology lab.
38
REFERENCES
  1. YavasY, SelubM. Intrauterine insemination (IUI) pregnancy outcome is enhanced by shorter intervals from semen collection to sperm wash, from sperm wash to IUI time, and from semen collection to IUI time. Fertil Steril 2004;82 (6): 1638–47.
  1. World Health Organisation laboratory manual for examination of human semen and sperm cervical mucus interaction, 4th Cambridge University press  1999.
  1. The 2008 Compendium of practice committee reports. Fertil Steril 2008; 9: 74–8.
  1. GluickDS, OverstreetJW, Factor LitwickP, et al. Sperm morphology, motility and concentration in fertile and infertile men. N Engl J Med 2001;345: 1388.
  1. KrugerTF, AcostaAA, SimmonsKF, et al. Predictive value of abnormal sperm morphology in In Vitro fertilization. Fertil Steril 1988; 49: 112.
  1. LarsenL, ScheikeT, Peter BondeJ, et al. Computer assisted semen analysis parameters as predictors for fertility of men from the general population. Hum Reprod 2000; 15(7): 1562–7.
  1. De GeyterM, CoppersB, NeischlagE. The diagnostic accuracy of computer aided sperm motion analysis. Hum Reprod 1998; 13: 2512–20.

Setting up of an IUI Laboratory4

40The laboratories involved in different reproductive services are usually known as ART laboratories. It is the most important component for the success of any ART program. IUI laboratory is an ART laboratory with small dimensions. It is used to process, store semen samples for the purpose of insemination.
 
REGISTRATION AND ACCREDITATION
According to The Indian Council of Medical Research (ICMR), registration of IUI laboratories is mandatory under appropriate licensing authority. In most of the states, the committees’ responsible for the implementation and supervision of PNDT act have the power to register and supervise ART labs also.
The ICMR guidelines1 have categorized the infertility clinics into four groups as follows:
  • Level-1A infertility clinics that do not handle gametes that have been donated, collected or processed in vitro. They can perform IUI with samples obtained from another registered semen bank or IUI laboratory.
  • Level-1B provides services for semen preparation, and basic semenology.
  • Level-2 and Level-3 provide all types of infertility services including IVF and ICSI. Additionally, Level-3 can conduct research involving gametes and embryos.
All units need to be registered except for level 1A. They also have to submit regular reports on all procedures performed (Fig. 4.1).
41
Fig. 4.1: IUI room
 
Laboratory Design (Figs 4.2A and B)
There are two important issues—IUI lab is an ART lab and maintenance of sterility is an uncompromising issue. General design and management of IUI lab is similar to any ART lab. Further growth and expansion of the laboratory should be reflected in the planning. Adequate space should be there for the following:
Storage space–
  • For the storage of liquid nitrogen cylinders and tanks
  • For disposables, media and other supplies
  • For keeping records
Sample collection room—It should have an attached toilet, bed and suitable aids to help in collection.
Procedure room—It should have all the facilities required for gynecological examination.
42
Fig. 4.2A: IUI lab
Fig. 4.2B: Laminar flow
43
 
Bench Space
Generally, it should be in the region of 100 to 200 square feet. It is preferable to have the laboratory adjacent to or near the IUI room, where the actual insemination is done.
 
Water
A water source is required to wash hands. It should be located in a corner and should be deep enough to avoid splashing.
 
Air
It should be clean, free of dust, smoke, gases and microbes.
It is important to take the following precautions:
  • It should be away from traffic, dust and fumes.
  • All electrical wiring should be concealed. There should be continuous power supply with power protection.
  • Avoid false ceiling.
  • Split or central air conditioning is preferable to window air conditioner. The laboratory should not get contaminated when the AC is being serviced or repaired.
  • Walls made up of joint free stainless steel and covered with epoxy paint are ideal. It should reach the roof and not be just half partitions.
  • Floor should be dust, crevices and crack free. It should be covered with large marble tiles, or granite tiles. Alternately one can also prepare the floor with vitrified tiles.
  • UV light is necessary to create a sterile area in the lab. It is important not to run the UV light when work is going on. One can run the light on the weekends when there is no work.
    44
  • One can use pressure modules to create positive pressure in the lab if in future one wants to convert it into an IVF lab.
  • All individuals handling samples in the lab should be screened for infections. It is advisable to change into sterile clothes, cap, mask and slippers while working in the lab. It is preferable to have clothes made up of cotton or silk and not of linen to avoid shedding of small threads.
 
EQUIPMENTS
 
Centrifuge (Figs 4.3A and B)
This should have a timer (up to 30 minutes) and a rotor (up to 3000 rpm) whose speed can be controlled. It is better to go for swing out rotor head.
Fig. 4.3A: Centrifuge with digital speed indicator and timer
45
Fig. 4.3B: Open centrifuge
 
Incubator (Fig. 4.4)
Plain incubators set at 37ºC temperature, with stainless steel interiors and uniform heating is required. Use of CO2 incubators is optional. The choice of media depends on the type of incubator available.
 
Laminar Flow Hood
It ensures a clean air inside the lab. It can be either a vertical or horizontal flow. Vertical flow is less harmful to the lab workers as the air flow is not directly aimed at their faces. On the other hand, a horizontal flow is more effective in reducing contamination. For IUI lab one can order for a one and a half feet breadth laminar flow. It is best to ask for a stainless steel top which is durable, as well as easier to clean and maintain (Fig. 4.5).
46
Fig. 4.4: Incubator
Fig. 4.5: 2 × 2 laminar air flow
47
 
Microscope
Binocular microscope preferably phase contrast is required to test the semen. It should have 10x, 40x, 100x objective lenses and a10× eyepiece (Fig. 4.6).
 
Digital Heater (Fig. 4.7)
It can be used as an alternative to plain incubator, especially when HEPES buffered media are used instead of bicarbonate based media. It is a cheaper alternative and gives excellent results.
 
Sperm Counting Chamber
Manual sperm analysis systems: Nondisposable chambers as Neubaur chamber or Makler chamber are commonly used. Makler chamber is preferred as it is very accurate. Nowadays, disposable sperm counting slides are also available.
Fig. 4.6: Microscope
48
Fig. 4.7: Digital heating block
One slide can be used for analyzing two samples (Fig. 4.8).
Automatic sperm analyzer systems: Systems such as CASA are used in labs with a large volume of work.
Fig. 4.8: Makler chamber
49
 
Refrigerator
It is used to store various chemicals and culture media. It should not be used for any other activity.
 
MEDIA (Fig. 4.9)
Majority of IUI and ART labs use readymade culture media. These are easily available, nontoxic, tested for quality control and have consistent performance. The commonly used media are as follows:
  • HEPES buffered culture media: These are simple balanced salt solutions or complex solutions supplemented with proteins, antibiotics and HEPES. When exposed to the atmosphere, HEPES maintains the pH between 7.2–7.4. Hence, these media do not require CO2 incubator to maintain the pH.
    Fig. 4.9: Media
    50
  • Bicarbonate based culture media: These media are simple balanced salt solutions or complex solutions supplemented with proteins, antibiotics and bicarbonate buffer. It requires a 5% CO2 atmosphere to maintain the pH between 7.2–7.4. These are useful for maintaining long term culture conditions, needed for IVF.
For short-term procedures as semen washing and processing, use of HEPES buffered media along with the use of simple digital heaters or incubators is adequate.
 
RECORD MAINTENANCE
Record of all the procedures, use of donor semen, details of semen analysis, type of method used for semen processing, consent of the couple should be maintained thoroughly (Figs 4.10 and 4.11).
Fig. 4.10A: IUI cannula with syringe
51
Fig. 4.10B: Disposables
Fig. 4.11: Instruments for IUI
52
IUI lab setup is simple. One must always plan for future expansion depending on estimated workload (Table 4.1). If the center has the plans to provide ART facilities in future, then IVF lab should be planned at this stage.
53
Fig. 4.12: Liquid nitrogen containers
54
REFERENCES
  1. National guidelines for accreditation, supervision and regulation of ART clinics in India. Indian Council of medical research, national academy of medical sciences (India),  New Delhi 2005; 49–57.

Ovulation Induction in IUI5

56IUI is one of the safest, least invasive and cost effective forms of assisted reproduction. Ovulation induction is an important step in this process, as it aims at stimulating maturation of more than one oocyte at a time, which improves the chances of fertilization and pregnancy.
 
RATIONALE FOR INDUCING OVULATION WHILE CARRYING OUT IUI
Ovarian stimulation has been shown to significantly improve the outcome in IUI cycles. Ovarian stimulation may improve the results of IUI by following two mechanisms:
  • By increasing the number of eggs available for fertilization
  • By overcoming a subtle defect in ovulatory function and luteal phase
Numerous studies have highlighted the benefits of ovarian stimulation with IUI. From a retrospective analysis of 45 studies, Guzick et al concluded that the combined pregnancy rates of ovulation induction with IUI were better (8.3–17.1%) than isolated ovulation induction without IUI or controlled ovarian hyperstimulation (COH)1.
 
OVARIAN STIMULATION OR INDUCTION
Patients requiring ovarian stimulation or induction can be categorized into two groups.
 
Ovulatory Patients (Ovarian Stimulation)
In these patients there is an established ovulatory pattern. Multiple studies have shown improved pregnancy rates 57with ovarian stimulation in these patients as compared to nonstimulated natural cycles.
 
Anovulatory Patients (Ovarian Induction)
These comprise about 20 to 30 percent of all female factor infertility.
Anovulatory patients are further divided by WHO into 3 categories:
  • Group I: Hypogonadotrophic hypogonadism (Hypothalamic Pituitary Failure)
  • Group II: PCOS (Hypothalamic Pituitary Dysfunction)
  • Group III: Ovarian failure
The aim of ovarian stimulation in ovulatory patients is to bring about multiple follicular development in order to increase the number of eggs produced and hence the number of embryos potentially available for implantation. When anovulation is the cause of infertility, ovarian stimulation is aimed at achieving monofollicular response.
 
Tests Before Ovarian Stimulation
A number of tests help in predicting the ovarian response to external stimulation and thus helps in determining the dosage of gonadotrophins. Out of these, basal FSH and ultrasound estimation of Antral follicle count (AFC) are the ones most commonly used. A basal FSH of >10 mIU/ml indicates a low ovarian reserve and a higher stimulation dose will be required. A basal antral follicle count, done between D2-D4 of cycle helps to establish the number of primordial follicles and is a good predictor of ovarian reserve. Other tests which can be done include 58ovarian volume, serum AMH, and serum inhibin. It is also important to check the tubal patency by hysterosalpingogram (HSG) or laparoscopy before the ovulation induction and IUI.
 
VARIOUS DRUGS COMMONLY USED FOR OVULATION INDUCTION
  1. Clomiphene citrate (CC)
  2. Letrozole
  3. Gonadotropins
  4. Clomiphene with Gonadotropins
  5. Letrozole with gonadotropins
  6. Gonadotropins with GnRH analogues
  7. Gonadotropins with GnRH antagonists
  8. CC/Letrozole with FSH with GnRH antagonist (the soft protocol)
 
Clomiphene Citrate (CC)
Clomiphene citrate is a selective estrogen receptor modulator with a structure that allows it to bind to hypothalamic estrogen receptors, this interferes with estrogen receptor replenishment in the hypothalamus, resulting in increased pituitary release of FSH. Increased FSH release drives folliculogenesis at the level of ovary.2 It is simple to use, cost effective and associated with fewer complications. It is the drug of choice for inducing ovulation in women with oligo-ovulatory and anovulatory cycles. In such patients, it has been reported to induce ovulation in 70–80% women with pregnancy rates varying from 30–50%.3 In combination with IUI, it has been successfully used in ovulating patients also.
59
Clomiphene citrate is administered in dosage varying from 50 to 250 mg per day for 5 days starting from the early follicular phase that is second to fifth day of cycle. The dose of CC is increased by 50 mg per cycle till ovulation is achieved. However, it is seen that cumulative conception rate does not increase beyond 150 mg dosage, as the antiestrogenic properties of CC manifest more with greater dosage and in high doses, CC interferes with implantation and pregnancy. We recommend a dose of 100 mg per day for five days.
 
Outcome After Clomiphene Citrate
Pregnancy
30%
Failure (no pregnancy despite ovulation)
40%
Resistance: no ovulation
25%
Antiestrogenic effect
5%
60
 
Letrozole*
Letrozole is an aromatase inhibitor which is now commonly used for ovulation induction. Aromatase is a member of cytochrome P450 enzyme complex which catalyzes the final rate limiting step in production of estrogen. It is required for hydroxylation of androstenedione to estrone and testosterone to estrogen. Letrozole acting as an aromatase inhibitor decreases the estrogen production. The reduced estrogen level causes release of estrogen negative feedback at the hypothalamus and pituitary resulting in increased gonadotropin production and stimulation of ovarian follicles.4 Unlike clomiphene citrate, letrozole does not have any effect on endometrium or cervical mucus because of absence of estrogen receptor depletion and its short half-life.
 
Letrozole and Ovulation Induction
The use of aromatase inhibitors for ovarian stimulation is indeed promising, especially in patients who have failed to respond to clomiphene, either because of clomiphene resistance or thin endometrium. These patients have responded well to aromatase inhibitors, and it is associated with good ovulation rates, thicker endometrium, and considerable pregnancy rates.5,6 Letrozole is associated with thicker endometrium and increased stromal blood flow, thereby providing a better uterine environment more favorable for implantation. Compared to clomiphene, letrozole has been shown to have higher pregnancy rates.
61
A number of randomized controlled trials have shown that Letrozole is comparable to gonadotropin with regard to pregnancy rates in IUI cycles, with significantly lower cost and better patient compliance.
Addition of letrozole to gonadotropin stimulation protocols decreases the gonadotropin requirement and increases the number of preovulatory follicles.7,8 Especially in patients who are poor responders, aromatase inhibitor decreases the negative feedback on FSH secretion and increase ovarian sensitivity to FSH.
 
Doses
It is administered in dose of 2.5–5 mg/day for 5 days starting from day 3 of the cycle. Few clinicians have used a single dose of 20 mg on day 3. However, the information regarding single dose is still limited. The single dose may be beneficial by causing maximal estrogen suppression early in the cycle and of early clearance.
 
Extended Letrozole Therapy
In a recent study conducted by Badawy et al, extended letrozole therapy (2.5 mg daily from day 1 of menses for 10 days) was used for CC resistant PCOS women. They observed that the long letrozole protocol (10 days) can produce more mature follicles and subsequently more pregnancies than the short letrozole therapy (5 days).9
 
Letrozole Step-up Protocol
Recently a new protocol known as letrozole step-up protocol has been reported by Mitwally et al. In this protocol, letrozole was administered in the step up doses consisting of one, two, three, and four tablets of letrozole 62(2.5 mg) daily on menstrual cycle days 2, 3, 4 and 5 respectively. When compared with standard clomiphene citrate protocol (CC 100 mg for 5 days starting on day 3), the step up letrozole protocol was associated with multifollicular development and a higher clinical pregnancy rates/treatment cycle (27.3% vs 11.8%).10 Extends FSH window by prolonging the suppression of estrogen levels by:
  • Preventing rising estrogen from suppressing endogenous FSH.
  • Control break-through estrogen production due to proliferation of granulosa cells.
  • Increases the duration of elevated FSH, resulting in multifollicular development.
 
Gonadotropins
Gonadotropins are usually indicated for patients in whom both Clomiphene citrate and Letrozole have failed to induce ovulation. Gonadotropins are the drugs of choice in WHO Group 1 patients (Hypogonadotrophic hypogonadism). They yield high pregnancy rate (17%) but their use is plagued by a high incidence of multiple gestation (30–40%). A recent Cochrane review (2007) on the available results of gonadotropins suggested that gonadotropins might be the most effective drugs when IUI is combined with controlled ovarian hyperstimulation.11
 
Choice of Gonadotropins
The choice of gonadotropin to be used depends upon the day 2 plasma LH/FSH/E2 levels. If serum LH is elevated, FSH containing gonadotropins are indicated, whereas if serum FSH is elevated (>10 mIU/ml), a combination of 63LH and FSH is used for ovarian stimulation. For ovarian stimulation in patients with hypogonadotropic hypogonadism, a combination of LH and FSH is used.
 
Factors Influencing the Dose of Gonadotropins
  1. BMI: Dosage is directly proportional to patient's BMI
  2. Ovarian reserve: FSH level above 10 IU/L indicates a need for higher dose.
  3. Age of the patient: Patients above 35 years of age need higher dose.
  4. Cause of infertility: Patients with PCOS need lower dosage whereas patients of unexplained infertility and hypogonadotropic hypogonadism need higher doses.
  5. Dose needed for stimulation in previous cycle.
 
Regimens
Three different regimes of gonadotropins are used for ovulation induction. They include:
  • Conventional regimen
    This regime has been used with success in clomiphene resistant and clomiphene failure cases. In this protocol, a daily dose of 75–150 IU of gonadotropins is started from day 2 or day 3. Serial USG for follicular monitoring and Serum Estradiol (E2) is performed from day 8 onwards. This protocol has yielded acceptable pregnancy rates of up to 30%.
  • Low dose step-up regime
    This regimen has especially been useful in women with PCOS. The principle behind this regimen is to find the “threshold“ level of FSH which will lead to the development of a single preovulatory follicle. The key feature of 64this regimen is the low starting dose (37.5–75 units/day) of drug, and a step-wise increase in subsequent doses, if necessary with an aim of achieving the development of a single dominant follicle rather than the development of many large follicles, so as to avoid the complications of OHSS and multiple pregnancy.12 Serum E2 level is measured and USG is performed on day 7 If day 8 Serum E2 is > 200 pg/ml or follicle size is above 10 mm, the same dose is continued. If, however, serum E2 level or the follicle size is inadequate, the dose is increased by 37.5 units/day every week till serum E2 level rises adequately.
    Low dose step-up regimen is a popular regimen for ovulation induction, as it is associated with acceptable success rate and decreased incidence of complications. However, this therapeutic approach is very unphysiological. Low dose regimen results in elevated levels of FSH during late follicular phase contrary to the natural cycles.
  • Step down regime
    The step down regime mimics the hormonal pattern in normally ovulatory women and induces development of one follicle at a time in anovulatory women. In a natural 65cycle, FSH promotes growth because of two events,” the FSH threshold” and “FSH window”. FSH threshold is the level of FSH below which no follicular growth can be initiated. The FSH window is the number of days that FSH levels are above the threshold, which accounts for the total number of follicles that are activated. Since sensitivity of follicle increases with development, the required FSH for a follicle will decrease. Balance between the decreasing levels of FSH and increasing FSH sensitivity is responsible for the growth of the dominant follicle and atresia of remaining follicles.
In a step down protocol HMG/FSH therapy at a daily dose of 150 units is started on day 2 and continued till a dominant follicle of > 10 mm is observed on TVS. After this, the dose is decreased to 112.5 units IM per day for 3 days, followed by 75 units IM per day for next 3 days. This dose is then continued till the day of hCG injection. Rest of the regimen is same as in the step-up regime.
 
Clomiphene Citrate with Gonadotropins
Sequential use of CC and gonadotropin (HMG or FSH) therapy has become an increasingly utilized method for COH for patients who fail CC therapy. In this protocol, CC 100 mg is administered from day 2 to day 6 and Injection FSH/HMG 75/150 units is given on day 6 and day 8. Transvaginal sonography is done from day 8 onwards and in case the follicle growth or number is inadequate, additional FSH/HMG injections are administered.
A combination of clomiphene citrate with gonadotropins has following advantages:
66
  1. Higher pregnancy rate than with CC alone.13,14
  2. More cost effective, as the dosage of gonadotropins is reduced.14,15
  3. Lesser multiple pregnancy rate than with gonadotropins alone.
  4. Lower incidence of OHSS, as compared to the conventional regime.
 
Disadvantage
The disadvantage of adding CC has been its antiestrogenic effect which has an adverse pregnancy outcome.
Effect of Etiology on PR in IUI
Indications
Bourne hall
Babies and US
Martinez
Indications
12.3%
14%
18%
Cervical
16%
18%
14%
Immunological
10%
9%
10%
Male subfertility (total motile >5 million)
21%
19%
10%
Donor
20%
Endometriosis
12%
11%
Ejaculatory fail
13.3%
14%
11%
 
Letrozole with Gonadotropins
Aromatase inhibitors are also being used in combination with gonadotropins, in order to reduce the requirement of gonadotropins and the side effects of high dose gonadotropin therapy. It appears to be a good alternative to CC in patients with unexplained infertility undergoing gonadotropin stimulated COH cycles combined with IUI therapy.
67
In a prospective nonrandomized study by Mitwally and Casper it was shown that aromatase inhibition with letrozole reduced the dosage of FSH required for COH without any undesirable antiestrogenic effects, which are commonly observed with use of CC in combination with gonadotropins. The pregnancy rate achieved was also significantly lower in the CC + FSH group (10.5%) compared with the letrozole + FSH group (19.1%) and FSH only group (18.7%).16
 
GnRH Analogue in Combination with Gonadotropins
In almost 15–20% of cycles, which have been stimulated with gonadotropins or CC, the exaggerated estradiol level due to the multifollicular development often provokes higher LH levels during the follicular phase or an untimely LH hormone surge, which leads to cycle cancellation. 68Therefore, in order to avoid interference from endogenous gonadotropin secretion, a combination of gonadotropins and GnRH analogs has being used for ovulation induction. Although GnRH analogs are routinely used in IVF cycles, their routine use in IUI cycles is not recommended. Recent Cochrane review has concluded that GnRH analogs do not significantly improve pregnancy rates in IUI.
 
GnRH Antagonists
GnRH antagonists act by competitive inhibition of GnRH receptors, which results in rapid decline in FSH/LH levels thus preventing premature LH surge. The drug can be given in a single dose or daily dose regimen.
The two protocols for administering GnRH antagonists are:
  1. Lubeck protocol: Gonadotropins are started as usual and antagonist is started when the follicle reaches a size of 14 mm, or from from 6th day of stimulation onwards in a dose of 0.25 mg/day till the day of hCG injection.17
  2. French protocol: Gonadotropins are started as usual and a single dose (3 mg) of antagonist is given when serum E2 level is about 150–200 pg/ml and follicular size is 14 mm.18
Both these protocols are equally effective in preventing premature LH surge. Their efficacy in preventing LH surge and pregnancy outcome has been compared to GnRH antagonist and is found to be equally effective. It has also been shown that administration of antagonists to patients having COH cycles with multifollicular development, significantly improves the pregnancy rates.
69
 
Advantages of Antagonist Protocol
  1. Use of antagonist allows the manipulation of follicular development so that IUI can be avoided at weekends without any detrimental effect on PR.
  2. When compared to agonist it is relatively simple and inexpensive. There is no suppression of estrogen and the effects are easily reversible.
  3. Antagonists are associated with lower rates of OHSS. The preserved pituitary response with antagonist has opened new paths in the treatment of patients at high risk of developing OHSS, as ovulation induction is possible by giving GnRH antagonist and so the deleterious effects of hCG are avoided.
70
REFERENCES
  1. GuzickDS, SullivanMW, AdamsonGD, CedarsMI, FalkRJ, PetersonEP, SteinkampfMP: Efficacy of treatment for unexplained infertility. Fertil Steril 1998;70 (2): 207.
  1. Practice Committee of the American Society of Reproductive Medicine. Use of clomiphene citrate in women. Fertil Steril 2004; 82: S90–6.
  1. HomburgR. Clomiphene Citrate—the end of an era? A mini review. Human Reprod 2005; 20: 2043–51.

  1. 71 MitwallyM. Casper R. Aromatase inhibitors in ovulation induction. Semin Reprod Med 2004; 22: 61–78.
  1. MitwallyMF, CasperRF. Aromatase inhibition: a novel method of ovulation induction in women with polycystic ovarian syndrome. Reprod Technol 2000; 10: 244–7.
  1. MitwallyMF, CasperRF. Use of an aromatase inhibitor for induction of ovulation in patients with an inadequate response to clomiphene citrate. Fertil Steril 2001; 75: 305–9.
  1. HealeyS, TanSL, TulandiT, BiljanM. Effects of letrozole on superovulation with gonadotropins in women undergoing intrauterine insemination. Fertil Steril 2004; 80: 1325–9.
  1. MitwallyM, CasperR. Aromatase inhibition reduces the dose of gonadotropin required for controlled ovarian hyperstimulation. J Soc Gynecol Investig 2004; 11: 406–15.
  1. BadawyA, MosbahA, TharwatA, EidM. Extended letrozole therapy for ovulation induction in clomiphene-resistant women with polycystic ovary syndrome: a novel protocol. Fertil Steril 2009; 92(1): 236–9.
  1. MitwallyMF, SaidT, GalalA, et al. Letrozole step-up protocol: a sucessful superovulation protocol. Fertil Steril 2008; 89, S23–4.
  1. CohlenBJ, HeinemanMJ. Ovarian stimulation protocols (antiestrogens, gonadotrophins with and without GnRH agonist/antagonist) for intrauterine insemination in women with subfertility. Cochrane Database systematic review. 2007 Apr 18;(2):CD005356
  1. MathurR, KailasamC, JenkinsJ. Review of the evidence base of strategies to prevent ovarian hyperstimulation syndrome. Hum Fertil 2007; 10(2): 75–85.
  1. LuPY, ChenAL, AtkinsonEJ, LeeSH, EricksonLD, OrySJ. Minimal stimulation achieves pregnancy rates comparable to human menopausal gonadotropins in the treatment of infertility. Fertil Steril 1996; 65: 583–7.
  1. KemmannE, JonesJR. Sequential clomiphene citrate –menotrophin therapy for induction or enhancement of ovulation. Fertil Steril 1983; 39: 772–9.

  1. 72 DickeyRP, OlarTT, TaylorSN, CuroleDN, RyePH. Sequential clomiphene citrate and human menopausal gonadotrophin for ovulation induction: comparison to clomiphene citrate alone and human menopausal gonadotrophin alone. Human Reprod 1993; 8: 56–9.
  1. MitwallyMFM, CasperRF. Aromatase inhibition reduces gonadotrophin dose required for controlled ovarian stimulation in women with unexplained infertility. Hum Reprod 2003; 18: 1588–97.
  1. DiedrichK, DiedrichC, SantosE, ZollC, Al-HasaniS, ReissmannT, et al. Suppression of the endogenous luteinizing hormone surge by the gonadotrophin-releasing hormone antagonist Cetrorelix during ovarian stimulation. Hum Reprod 1994; 9: 788–91.
  1. OlivennesF, FanchinR, BouchardP, de ZieglerD, TaiebJ, SelvaJ, et al. The single or dual administration of the gonadotropin-releasing hormone antagonist cetrorelix in an in vitro fertilization–embryo transfer program. Fertil Steril 1994; 62: 468.

Monitoring in Intrauterine Insemination6

74Intrauterine insemination (IUI) is an artificial insemination technique used for couples with unexplained infertility, minimal male factor infertility, and women with cervical mucus problems. It is the process by which sperm is placed into a female's reproductive tract using artificial means, rather than by natural sex. IUI can be performed using the husband's sperm or donor sperm. Before insemination, sperm is prepared for IUI, a process that involves washing and separation. Sperm washing cleanses the sperm of potentially toxic chemicals which may cause adverse reactions in the uterus. Separation selects out motile sperms and concentrates them into a small volume.
IUI is often done in conjunction with ovulation-stimulating drugs, such as clomiphene citrate, gonadotropins, or urofollitropins. Insemination is performed at the time of ovulation, usually within 24–36 hours after the LH surge is detected, or after the “trigger” injection of hCG is administered. Especially if injectable ovulation stimulating drugs are used in an IUI cycle, careful monitoring is essential. Monitoring includes periodic blood tests and ultrasound assessment beginning around day 6 of the woman's cycle. Results of these tests will indicate when the eggs are mature, prompting the hCG shot.
 
MONITORING OF FOLLICULAR GROWTH AND ENDOMETRIAL DEVELOPMENT
Ovulation induction is undertaken to stimulate the ovaries to produce 1–2 mature oocytes per cycle. Combined with IUI, ovulation induction increases the chances of pregnancy. However this is associated with an inherent risk of hyperstimulation1 and multiple pregnancy.2 Therefore, it 75is important to monitor these cycles regularly. There are three reasons for monitoring ovarian stimulation:
  • To evaluate if the dose of the stimulating drug being used is optimal.
  • To find the optimal time for inducing ovulation with human chorionic gonadotropin.
  • To avoid excessive stimulation which may cause ovarian hyperstimulation syndrome (OHSS).
The follicular growth can be monitored with serial transvaginal ultrasonography, serial serum estradiol level estimation and LH assays to detect LH surge.
 
Ultrasonographic Monitoring
Serial ultrasonography (USG) is the most important tool for monitoring induced ovulation cycles. Initially, transabdominal sonography was used for monitoring stimulated cycles. However, nowadays transvaginal sonography (TVS) has become the most important tool for monitoring stimulated cycles. TVS is a noninvasive method to measure follicular size and endometrial thickness.
 
Monitoring Follicular Growth
TVS provides a direct assessment of follicular development. It provides valuable information about both number as well as the size of the follicle. The follicular size is measured by taking mean of 2 or 3 largest perpendicular diameters of each follicle. All follicles more than 10 mm in size are taken as significant and their size is measured and recorded.3
A baseline scan is performed on day 2 of the cycle to exclude the presence of ovarian cysts ≥ 3 cm and other 76pathologies such as PCOS, polyps, endometriosis or hydrosalpinx. Serial ultrasound scanning for monitoring follicular growth is started from day 7 or 8 onwards. The follicles normally grow at the rate of 2–3 mm a day, once the leading follicle reaches 15–16 mm size.4 Serial ultrasound helps us to determine the exact time for triggering ovulation, especially in stimulated cycles.
 
Assessing the Follicular Maturity
Researchers in a number of studies5,6 have tried to predict the follicular size which would contain mature oocytes; however, a definitive size of follicle which confirms the maturity of oocytes is still controversial. A follicle measuring 18–20 mm has often been found to contain a mature oocyte. Therefore, a mean follicular diameter of 18–20 mm has been arbitrarily set as the cut-off level for inducing ovulation.
 
Predicting the Risk of OHSS and Multiple Births
The number of follicles can also predict whether a particular patient would be susceptible to ovarian hyperstimulation syndrome (OHSS). Age, number of preovulatory follicles and E2 levels, also affect the incidence of multiple births. Recommendations to prevent multiple pregnancies have been varied and include withholding human chorionic gonadotropin (hCG) administration when more than six follicles are ≥12 mm in diameter7; when more than three follicles are ≥14 mm8,9 or ≥16 mm in diameter10 and when more than two11 or three12,13 follicles are ≥18 mm in diameter. A recent study has proposed withholding hCG when there are seven or more preovulatory follicles of diameter ≥10 mm.
77
If there are more than 4 follicles larger than 16 mm size or more than 8 follicles larger than 12 mm size, it is best not to give hCG, as it can cause OHSS and may lead to high order multiple births.
In case of doubt, it is best to measure serum estradiol (E2) level. If the E2 level is less than 1500 pg/ml, hCG may be given. However, if the E2 level is more than 1500 pg/ml, hCG should rather be avoided.
 
Monitoring the Endometrium
USG is also used to assess the endometrium for its thickness and reflectivity (appearance). Shoham et al have demonstrated that no pregnancy occurs if endometrium is < 7 mm in thickness. Endometrial thickness of 7–8 mm indicates endometrial maturity required for pregnancy.14
Ultrasound is also used to study the echogenicity of the endometrium.15 Three echo patterns have been described:
  1. Hypoechogenic endometrium is present in the early follicular phase.
  2. Triple line endometrium is seen in the late follicular phase (Fig. 6.1)
  3. Hyperechogenic endometrium is seen in the luteal phase.
A triple line endometrium with a thickness of more than 7–8 mm is best conducive to pregnancy.
Color Doppler ultrasonography has also been suggested for studying uterine and ovarian blood flow and endometrial peristalsis after IUI in order to improve and predict success after IUI. However, it has not been shown to be of any practical significance.
78
Fig. 6.1: USG showing triple line endometrium
 
Serial Serum Estradiol Levels
Plasma estradiol correlates closely with the stage of development of the dominant follicle in a natural cycle. But, this is not true for the stimulated cycles, as serum estradiol reflects the total output of all developing follicle irrespective of their sizes. Another problem of serum estradiol estimation is the inconvenience of blood testing faced by the patient, both in terms of disruption of day to day routine, as well as costs. Therefore, ultrasound monitoring has replaced estradiol monitoring in most centers.
Practically, serum estradiol level is measured in pure gonadotropin stimulated cycles with or without 79GnRH analog on day 8 of stimulation, to assess follicular response. Serum estradiol levels > 200 pg/ml indicates adequate dosage of gonadotropins.
It is also indicated when ultrasound detects more than four follicles of an average size > 16 mm or more than 8 follicles of an average size > 12 mm on the day of ovarian stimulation. In this situation, an estradiol level of > 1500 to 2000 pg/ml would indicate a risk of OHSS and hence, withholding of ovulation trigger and cancellation of the cycle. If however, serum estradiol level is < 1500 pg/ ml, one can use a GnRH analog to trigger ovulation, provided that the ovarian stimulation is not in a down regulated cycle.
 
Detecting the LH Surge
In natural cycles, the LH surge acts as the trigger for ovulation. Therefore it can be used to predict ovulation. Studies have indicated that in clomiphene stimulated cycles, LH surge monitoring is as good as hCG for timing of IUI.16 Both urinary and blood tests are available to estimate the LH levels and hence to detect the LH surge. A blood level of > 10 IU/L is said to correlate with the LH surge. However, the accuracy of the urinary LH test has been questioned because false-negative results occur frequently when peak levels are < 40 IU/L, when women have surges lasting >10 hours in duration, or when diluted urine is tested17. The study by Lloyd et al18 showed that when LH kits alone were used to time IUI, 36% of inseminations were timed incorrectly and 15% of women had already ovulated.
80
In patients who are undergoing ovarian stimulation and are not using GnRH analogs for down regulation, there remains a possibility of premature endogenous LH surge prior to the administration of hCG to bring about ovulation. In case of a premature LH surge, ovulation will occur in relation to the surge, rather than occurring in relation with the hCG injection. In such conditions IUI will have to be planned as per the LH based assessment of ovulation. The incidence of premature LH surges in these patients has been studied in many studies. Cohen et al19, in a randomized controlled study, detected endogenous LH surges in 24% of the stimulated cycles. Other researchers have reported premature surge in up to 33% of the HMG stimulated cycles20.
Serum or urinary LH levels should be measured once or twice daily, after the leading follicle reaches a diameter of 14 mm. If an LH surge is detected, Injection hCG 10,000 units should be given immediately, and should be followed by an IUI on the same day. A repeat insemination is performed the next day. The hCG injection is required to supplement the LH secreted by the body, which is not adequate enough to induce the necessary maturational changes in all oocytes, if more than one follicle in the ovary gets stimulated.
Premature LH surges in stimulated cycles have a negative impact on pregnancy outcome.16 Therefore; treatment strategies to prevent such premature LH surges are required in order to improve the outcome of IUI in these patients.
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Ovulation Trigger and Timing of Insemination
The end point of any ovulation induction protocol is to identify the best time for triggering ovulation. The probability of conception seems to correlate directly to the number of follicles >11 mm at the time of triggering ovulation.21. All follicles > 10 mm should be documented.
The ovulation is triggered when the following conditions are met:
  1. Leading follicle is 18–20 mm in diameter.
  2. The number of follicles more than 16 mm is not more than 4 or that of follicles more than 12 mm is not more than 8.
  3. Serum E2 is not more than 1500 to 2000 pg/ml (Serum Estradiol is done only if the total number of follicles exceed 12 and there is a potential chance of OHSS).
  4. There is no LH surge as seen by testing the morning sample of urine.
Various ovulation triggers available include:
  1. Human chorionic gonadotropin (hCG): hCG is the most commonly used drug for inducing the final maturation of the follicle, and ovulation. The mid-cycle LH surge is essential for normal oocyte maturation and ovulation. The B-subunit of hCG confers biological activity and displays up to 80% homology with LH. Partially purified urinary hCG preparations have been used for decades as a surrogate for LH to achieve final oocyte maturation and ovulation in controlled ovarian hyperstimulation (COH) protocols. This facilitates correct timing of IUI. Urinary derived hCG (uhCG) in a dose of 5000 IU or 10,000 IU/IM is given and IUI is 82done 34 to 36 hours after the hCG injection, in case of a single insemination.
    Recombinant hCG (rhCG) is also available for use in these cases. It is administered in a dose of 250 µg subcutaneously. Studies comparing uhCG and rhCG for ovulation induction have found both of them to be equally effective17, 21, 22 However, rhCG is associated with better patient tolerance and is associated with lesser side effects. Patients treated with rhCG have been shown to have a reduced incidence of OHSS.23
    The one disadvantage of hCG is that it may cause OHSS in some patients. Therefore, it is important to adhere strictly to the cancellation criteria mentioned above to decrease the incidence of OHSS in IUI cycles.
  2. Recombinant LH (rLH): Recombinant LH produces a more physiological LH surge to trigger the final follicular maturation and ovulation. It has a shorter half-life than hCG and results in a LH peak lasting for 48–60 hours, in contrast with a peak lasting 7 days with use of hCG. A recent systematic review compared uhCG, rhCG and rLH in this role and the authors concluded that all three are equally efficacious for inducing the final follicular maturation.18
  3. GnRH agonist: Gonadotropin releasing hormone (GnRH) induces surges of LH and FSH, with similar luteal phase length and progesterone levels as hCG cycles. GnRH has a shorter half-life (3–5 hours) and can be used as a substitute for hCG in ovulation induction cycles for women at risk of OHSS. Many randomized controlled 83trials (RCTs) have confirmed that agonist trigger can eliminate OHSS.18, 24 The standard GnRH regimen for ovulation induction includes: Injection Luprolide 500 µg subcutaneously, which is repeated after 12 hours. IUI is performed 36 hours after the first injection.
 
Case 1
A 30-year-old woman with primary unexplained infertility for 5 years, was for an IUI after initial basic investigations. She received 100 mg of clomiphene citrate from day 2 to day 6. USG on day 9 showed 2 dominant follicles. Serial scans were then performed and injection hCG 10,000 U was given when the leading follicle reached 18 mm. IUI was performed 36 hours later.
 
Case 2
A combination of Letrozole and FSH was used for stimulation in this case. USG on day 8 showed one dominant follicle on right side and two on left side.
84
REFERENCES
  1. McElhinneyB, MClureN. Ovarian hyperstimulation syndrome. Baillieres Best Practi Res Clin Obstet Gynecol 2000; 14: 103–22.
  1. JacobsHS, AgarwalR. Complications of ovarian hyperstimulation. Baillieres Best Practi Res Clin Obstet Gynecol 1998; 12: 565–79.
  1. Richard P.Dickey, Steven NTaylor, PeterY Lu, et al. Risk factors for high-order multiple pregnancy and multiple birth after controlled ovarian hyperstimulation. Results of 4,062 intrauterine insemination cycles. Fertil Steril 2005;83: 671–83.
  1. LeerenttveldRA, WladimiroffJW. 24-h echographic profile study on follicular growth prior to laparoscopic aspiration of graffian follicle in clomiphene citrate/human chorionic gonadotrophin stimulated cycle. Eur J Obstet Gynecol Reprod Biol 1986; 22: 287–91.
  1. HackeloerBJ. The ultrasonic demonstration of follicular development during the normal menstrual cycle and after hormone stimulation: KurjakA (Ed) Recent advances in ultrasound diagnosis. Amsterdam Excerpta Medica,  197: 122–8.

  1. 85 RitchieWG. Ultrasound in the evaluation of normal and induced ovulation. Fertil Steril 1985: 43:167–81.
  1. ValbuenaD, SimonC, RomeroJ, et al. Factors responsible for multiple pregnancies after ovarian stimulation and intrauterine insemination with gonadotropins. J Assist Reprod Genet 1996; 13: 663–8.
  1. PittrofRU, ShakerA, DeanN, et al. Success of intrauterine insemination using cryopreserved donor sperm is related to the age of the woman and the number of preovulatory follicles. J Assist Reprod Genet 1996; 13: 310–14.
  1. TakokoroN, VollenhovenB, ClarkS, et al. Cumulative pregnancy rates in couples with anovulatory infertility compared with unexplained infertility in an ovulation induction program. Hum Reprod 1997; 12: 1939–44.
  1. The treatment of infertility by the high intrauterine insemination of husband's washed spermatozoa. Hum Reprod 1988; 3: 939–43.
  1. ZikopoulosK, WestCP, ThongPW, et al. Homologous intrauterine insemination has no advantage over timed natural intercourse when used in combination with ovulation induction for the treatment of unexplained infertility. Hum Reprod 1993; 8: 563–7.
  1. TomlinsonMJ, Amissah-ArthurJB, ThompsonKA, et al. Prognostic indicators for intrauterine insemination (IUI): statistical model for IUI success. Hum Reprod 1996; 11: 1892–6.
  1. HughesEG, CollinsJA, GunbyJ. A randomized controlled trial of three low-dose gonadotropins protocols for unexplained infertility. Hum Reprod 1998; 13: 1527–31.
  1. ShohamZ, Di CarloC, PatelA, et al. Is it possible to run a successful ovulation program based on ultrasound monitoring? The importance of endometrial monitoring. Fertil Steril 1991; 56: 836–41.
  1. SmithB, PorteR, AhujaK, CraftI. Ultrasounic assessment of endometrial changes in stimulated cycles in an in vitro fertilization and embryo transfer program. J in vitro Fertil Embryo Transfer 1984; 1: 233–8.

  1. 86 IoannisP Kosmas, AthinaTatsioni, HumanMusavi Fatemi, et al. Human chorionic gonadotropin administration vs luteinizing hormone for intrauterine insemination timing, after administration of clomiphene citrate: a meta-analysis. Fertil Steril 2007;87: 607–12.
  1. The European Recombinant Human Study Group, EngrandP et al. Human recombinant luteinizing hormone is as effective, but safer than human chorionic gonadotrophin in inducing final follicular maturation and ovulation in IVF procedures: results of a multicentre double blind study. J Clin Endocrinol Metabol 2001;86: 2607–13.
  1. GnRH agonist for triggering final oocyte maturation in patients at risk of ovarian hyperstimulation syndrome: still a controversy? J Assist Reprod Genet 2008;25: 63–6.
  1. CohlenBJ, te VeldeER, van KooijRJ, et al. Controlled ovarian hyperstimulation and intrauterine insemination for treating male subfertility: a controlled study. Hum Reprod 1998; 12: 1553–8.
  1. ManziDL, DumezS, SottLB, NulsenJC. Selective use of Luprolide acetate in women undergoing super ovulation with intrauterine insemination results in significant improvement in pregnancy outcome. Fertil Steril 1995; 63: 866–73.
  1. Sakhel,M. Khedr,S. Schwark,M. et al. Comparison between urinary and recombinant hCG during ovulation induction in IUI cycles: a prospective randomized clinical trial. Fertil Steril 2004; 82: S23.
  1. MichaelLudwig, KevinJ Doody, KathleenM Doody. Use of recombinant hCG in ovulation induction. Fertil Steril 2003; 79: 1051–59.
  1. Al-InanyH, AboulgharMA, MansourRT, Proctor M Recombinant versus urinary gonadotrophins for triggering ovulation in assisted conception. Hum Reprod 2005; 20: 2061–73.
  1. DíazI, GuillénA, PachecoA, et al. Endocrine modifications associated with final oocyte maturation with gonadotropin-releasing hormone agonists’ vs human chorionic gonadotropin in women undergoing intrauterine insemination. J Reprod Med 2008; 53: 33–9.

Role of Doppler in Intrauterine Insemination7

88
 
INTRODUCTION
Success of intrauterine insemination (IUI), is based on selection of optimum stimulation protocol, correct timing of hCG before IUI, good semen preparation and optimum luteal support. Both stimulation protocol and timing of IUI, can be optimized by ultrasound assessment. It is done before starting the stimulation, that is day 2–3 scan and before planning human chorionic gonadotrophin (hCG) for IUI.
Using color Doppler in this assessment is mandatory because a large number of biochemical or hormonal changes occur during the cycle, and they reflect as vascular and morphological changes in the ovaries and uterus and vascular changes can be assessed by Doppler. 2D US gives information about the anatomical status and Doppler reveals about the functional status of ovary, follicle, uterus and endometrium.
 
Baseline Scan
Scan done between 2nd to 4th days of the cycle, is known as a baseline scan. At this time of the cycle, the ovaries have no active follicles or corpus luteum and estrogen and progesterone are both at lowest levels. Endometrium is thin like a single line as it has shed off during menstruation.
This scan consists of transvaginal sonography—TVS, transvaginal color Doppler—TVCD and volume USG of ovaries and uterus. It is done to predict the ovarian reserve and response which can guide to decide the stimulation protocols for ovulation induction. Uterus is scanned to diagnose abnormality and assess receptivity (Fig. 7.1).
89
Fig. 7.1: Ovarian stimulation protocols for ovulation induction
 
Baseline Scan of Ovaries
2D ultrasound assessment of the ovaries consists of assessment of ovarian diameters and volume and counting of antral follicles and qualitative assessment of stromal density. When scanning the ovary, the probe is rotated at various angles to find out the most longitudinal section of the ovary. To calculate, the volume of the ovary on 2D US a second section is taken by rotating the probe 90° from this most longitudinal section. On this section two perpendicular measurements are taken one perpendicular and one parallel to the ultrasound beam. Volume is calculated by applying the formula (x × y × z) × 0.53. Color or power Doppler are used to see the presence of vessels in the 90stroma. If vascularity is present, pulse Doppler is used for quantitative assessment of the flows —intraovarian resistance index (RI) and peak systolic velocity (PSV). Then 3D and 3D power Doppler volume of the ovary is acquired for ovarian volume and stromal volume, counting number of antral follicles. Calculation of ovarian volume by VOCAL (volume calculation by computer) on 3D US is much more accurate (Fig. 7.2). For VOCAL usually rotating angle of 30° is used but in case of an ovary which is not perfectly oval, angle of 15° may be preferred.
Global vascular indices VI (vascularity index), FI (flow index) and VFI (vascularity flow index) may be calculated. The values for these are not yet established but initial studies indicate that they are likely to give promising results (Figs 7.3A and B).
Fig. 7.2: Three-dimensional ultrasound with volume calculation of ovary by VOCAL
91
Figs 7.3A and B: (A) Low resistance ovarian stromal flow, (B) High resistance uterine artery flow
Predictors of ovarian response are1
  • Number of antral follicles
  • Ovarian stromal FI
  • Total ovarian stromal area
  • Total ovarian volume
92
In that order of importance. This means number of antral follicles, ovarian flow, RI (resistance index) and PSV (peak systolic velocity) and ovarian FI can be most routinely used to calculate the dose requirement of the patient for ovarian stimulation.
Antral follicle count is a better marker than basal FSH for selection of older patients with acceptable pregnancy rates.2 Antral follicle count and ovarian volume showed significant correlation with AMH, total testosterone and free androgen index.3
Antral follicles can be counted on 2D ultrasound by scanning across the whole ovary while counting the follicles. But this has high chances of error when follicles are multiple as in polycystic ovaries. In these cases, counting the antral follicles by 3D US is much more precise. After taking a 3D volume of the ovary and doing a VOCAL for it, the ovary is rendered in inversion mode. This clearly shows all the follicles as solid balls and by rotating them they can be counted fairly accurately. A more precise method is using Sono AVC (automatic volume calculation). Using this software each follicle is coded by a different color and diameters and volumes of each is calculated separately (Figs 7.4A and B). Intraobserver and interobserver reliability of automated antral follicle counts made using three-dimensional ultrasound and SonoAVC a preferred method4.
It has been shown by Zaidi et al. that stromal blood flow velocity after pituitary suppression was an independent predictor of ovarian response.5 Kupesic has shown correlation in the ovarian stromal flow index and number of mature oocytes retrieved in an IVF cycles and pregnancy rates.1
93
Figs 7.4A and B: (A) 3D volume of polycystic ovary rendered in inversion mode, (B) 3D volume of polycystic ovary with Sono AVC
94Intraovarian stromal RI on baseline scan of < 0.58 is suggestive of good ovarian response and RI > 0.68 suggests poor ovarian response. Intraovarian stromal PSV < 5 cms/sec suggests that higher doses of gonadotropins would be required for stimulation where as PSV> 10 cm/sec suggest high possibility of hyperresponse. By a complex combination of RI and PSV, the doses for ovulation induction can be calculated (Table 7.1).
 
Uterus
Baseline scan of the uterus is done to assess the uterocervical length by ultrasound on longitudinal section of the uterus. For intrauterine insemination (IUI), the uterocervical length is measured from the fundal end of the endometrium to the external os and is known as functional or physiological uterocervical length. (Fig. 7.5). Doppler of the uterine artery is done on this scan. Uterine artery RI > 0.79 indicates that high doses of stimulation will be required for endometrial maturation.6 Subendometrial flow on day 2 if is present indicates a low receptive endometrium for implantation. This is so because on day 2 ovaries are silent, estrogen levels are very low and therefore no endometrial vascularity is expected normally. If it is present, it is either high basal estrogen of polycystic ovarian syndrome or inflammation of the endometrium leading to increased vascularity.
95
Fig. 7.5: Measuring functional uterocervical length
 
Preovulatory Scan
A follicle that grows to 14 mm is a dominant follicle. It grows at a rate of 2–3 mm/day and ovulation occurs at 18–24 mm size usually.
A rounded 16–18 mm sized follicle with thin walls and no internal echogenicity is seen. A sonolucent halo appears surrounding the follicle 24 hours prior to ovulation. Cumulus oophorus a small projection from wall in the follicular lumen may be seen.
Follicular flow can be first detected when follicular size is 10 mm.7 A mature follicle on color Doppler shows vascularity surrounding at least 3/4th of the follicular circumference. On pulse Doppler these vessels show PSV >10 cm/sec. (Figs 7.6A to C). This means that if the follicle is said to be functionally mature when PSV is 10 cms/sec, that is the time when the LH surge starts and under the effect of that LH, the perifollicular PSV keeps on rising constantly.
96
Figs 7.6A to C: (A) Perifollicular flow on power Doppler, (B) Spectral (C) Doppler of perifollicular flow
Its resistance starts falling two days prior to ovulation and achieves RI 0.4–0.48,8 at maturity. This is the time when hCG is given routinely and IUI, is done after 36–38 hours later.
Follicular blood flow velocity starts increasing 12 hours after LH peak, approximately 24–26 hours before rupture and continues till 72 hours after rupture. The perifollicular PSV rises as high as 45 cm/sec before an hour of ovulation. We have done a study of 300 IUI cycles based on this to find out appropriate time of IUI based on pre-hCG 97perifollicular vascularity. Single IUI, was done between 36–38 hours and double IUI were done at 12–14 hours and 36–38 hours in all patients in whom perifollicular PSV >15 cm/sec. When perifollicular PSV on the day of hCG was more than 20 cm/sec, and perifollicular RI was within normal range, double IUI has given significantly higher pregnancy rates.9 This indicates that in patients with higher perifollicular pre-hCG, PSV, IUI done at 12–14 hours has been fruitful. Actually single IUI at 12–14 hours would suffice in patients in whom pre-hCG perifollicular PSV is more than 20 cm/sec (Fig. 7.7).
Fertilization of a follicle with a PSV of less than 10 cm/sec, has high chances of embryo with chromosomal abnormality.
Rising PSV with steady low RI suggests that the follicle is close to rupture.7 Steady or decreasing PSV with rising RI suggests that the follicle is proceeding towards LUF.10
Fig. 7.7: Graphic representation showing increased pregnancy rates with double IUI in patients with perifollicular PSV >20 cm/sec
98
 
Application of 3D US for Follicular Assessment
When there is multifollicular development as in PCO, the follicular shapes become polygonal, and therefore, follicular diameter may not be a reliable parameter. Follicular volume by 3D is a more reliable parameter. Follicular volumes of between 3–7 cc are optimum for oocyte retrieval. The limits of agreement between the volume of the follicular aspirate and 3D volume of the follicle were +0.96 to −0.43 with 3D and +3.47 to −2.42 by 2D volume estimation.11
Cumulus can be seen in more almost 90% of follicles using 3D US rendering. This is possible only in upto 40–45% follicles by 2D US. On the day of hCG, if cumulus like echo is not seen in all three planes in the follicle, it is less likely to be mature fertilizable oocyte.12
3D power Doppler gives idea about the global vascularity of the follicle. The 3D power Doppler indices give quantitative global assessment of vascularity. The indices are vascularity index (VI), that assesses the abundance of flow, flow index (FI), that assesses the average intensity of flow and vascularity flow index (VFI), that assesses the perfusion of the tissues in the volume. We have done a large study taking this into consideration. Based on study of > 2500 cycles, follicular volume of 3–7.5 cc, VI 6–20 and FI > 35 if used as additional features of a mature follicle apart from the follicular features mentioned previously, increased the pregnancy rates significantly13 (Table 7.2 and Figs 7.8A to D).
 
Features of a Mature Endometrium
On 2D ultrasound endometrium of 6 mm or more in thickness, preferably 8–10 mm that is triple line is good enough.
99
Figs 7.8A to D: (A) Cumulus oophorus in follicle on 3D US of follicle rendered view. (B) 3D power angiography of perifollicular flow. (C) 3D power Doppler volume of follicle with VOCAL and shell volume calculation. (D) Calculation of 3D power Doppler vascular indices (volume histogram) after VOCAL and shell volume for perifollicular flow
100
But its morphological assessment is essential. Because of orderly organization of glandular elements in proliferative phase, endometrium is generally hypoechoic in this phase, though the morphology can be graded as follows.
 
Endometrial Grading14 (Figs 7.9A to C)
  • Type A endometrium: Homogenous hyperechoic endome­trium.
  • Type B endometrium: Multilayered with intervening area more echogenic than myometrium.
  • Type C endometrium: Multilayered with intervening area hypoechoic to myometrium.
2D ultrasound assesses the anatomy of the endo­metrium but its functional maturity can be assessed by Doppler of spiral arteries.
Spiral arteries supply the endometrium and these are the vessels that undergo substantial changes during menstrual cycle. Estrogen causes vasodilatation and progesterone antagonizes the effect. Therefore, higher E2/P ratio is responsible for higher blood flow through uterine vascular bed.
101
Figs 7.9A to C: Grade A, B, and C endometrium
Thus, endometrial color Doppler gives direct information about estrogen and progesterone levels and in turn also about functional maturity of the follicle and endometrium.
 
On color Doppler, the vascularity of the endometrium is classified by Applebaum as follows (Figs 7.10A to D).15
  • Zone I: Blood vessels reaching myometrium surrounding the endometrium
  • Zone II: Blood vessels reaching hyperechoic endometrial edge
  • Zone III: Blood vessels reaching internal endometrial hypoechoic zone.
    102
    Figs 7.10A to D: Zones I, II, III and IV vascularity seen in endometrium on color and power Doppler
  • Zone IV: Blood vessels reaching endometrial cavity.
On pulse Doppler, these blood vessels should show RI 0.49–0.59 and PI 1.1–2.3.
Even if TVCD of follicle is normal, endometrial and uterine artery indices should be within normal limits for implantation. Absent subendometrial and intraendometrial vascularization on the day of hCG, appears to be a useful predictor of failure of implantation in IVF, irrespective of morphological appearance.16 Conception rates according to zones of vascualrity in two different studies are quoted as
• Zone I
3.5%–7.5%
5.2%
• Zone II
15.8%–29.7%
28.7%
• Zone III
24.2%–47.8%
52%
• Zone IV
67.3%
74%
103
It has also been observed that when pregnancy is achieved in absence of endometrial and subendometrial flow on the day of embryo transfer, more than half of these pregnancies will finish as spontaneous miscarriage.17
But spiral arteries have higher PSV and lower resistance in natural cycles as compared to stimulated cycles may it be clomiphene citrate or gonadotropins.
Apart from endometrium and follicle it is also essential to evaluate the uterine artery flow on the dominant side before hCG. Estrogen receptors are also present in uterine arteries and cause vasodilatation in these vessels also. The normal values of uterine artery indices are RI 0.60–0.80 and PI 2.22–3.1615. Embryo transfers in IVF cycles are also cancelled if the uterine artery PI >3.2.18 hCG administration induces significant increase in the resistance of uterine artery for 48 hours which can affect its evaluation on the day of follicular aspiration/rupture therefore, Doppler study for uterine receptivity should be done on the day of hCG.6 Moreover, the Doppler studies are better done at the same time every day as a circardian rhythm, is seen in uterine artery flow in periovulatory phase.
As for the follicle, the endometrium also if evaluated by 3D and 3D power Doppler for receptivity before hCG, gives better implantation rates. It gives endometrial volume which is considered to be a more reliable parameter than endometrial thickness. More over 3D power Doppler gives idea about quantitative global vascularity of the endometrium, instead of 2D Doppler where we can see vessels only in one plane at a time and also interrogate a few vessels not all by pulse Doppler. In our study,13 3D power Doppler indices that we have found reliable 104are endomentrial volume >3 cc, FI >20 and VFI >5 (Figs 7.11A and B). There are several other people who have also worked on endometrial receptivity assessment by 3D and 3D power Doppler. A study by Luis T Merce et al. of 40 IVF cycles has shown endometrial volume of 3–7 ml as most favorable for conception.
Figs 7.11A and B: 3D power angiography and volume histogram of endometrium
105Median values for a favorable endometrium is 4.28 +/−1.9 ml.19 Another study shows that endometrial volume of >2.0 ml has a significantly higher pregnancy rates and no pregnancies were recorded with endometrial volume of <1 ml.20
No pregnancy occurred when endometrial volume was <3 ml and VI <10. Exceptionally better pregnancy rates are achieved with endometrial volume >7 ml and subendometrial VI between 10–35%.30
A study by Kupesic et al shows lower resistance index of 0.49–0.57 in subendometrial vessels and FI of 11.0–15.4 in conception cycles as compared to 9.5–13.3 otherwise.21
VFI on the day of hCG is more sensitive than volume, VI and FI for prediction of pregnancy. VFI >0.24 has sensitivity of 83.3%; specificity of 88.9%; PPV 93.8%; NPV 72.3% for prediction of pregnancy with 33% pregnancy rate.22
Scoring system has also been developed for endometrial receptivity, which apart from the endometrial thickness, morphology, vascularity and uterine artery Doppler also includes uterine contraction count. Calculating contractions/min >5/min is considered bad for implantation and, 3 contractions/minute is considered good for implantation. Though, this scoring system is not practically applicable as even with full scores of the often used uterine scoring systems, pregnancy might not occur.
 
Secretory Scan
Corpus luteum is a cystic structure with thick shaggy walls and echogenecity in the lumen. But it is known to have variable appearances like ground glass echogenicity in lumen or lace like echogenecities. Secretory changes are 106seen in the endometrium in the form of echogenecity of the endometrium that starts from outside, proceeding to the central line making a ring sign of the endometrium.23
A clear correlation between RI of corpus luteum and plasma progesterone levels has been seen in natural cycle. RI of the corpus luteum can therefore, be used as an adjunct to plasma progesterone assay as an index of luteal function.24
A healthy corpus luteum will show a vascular ring on color Doppler and on pulse Doppler these vessels show RI 0.35–0.50, PI 0.70–0.80 and PSV 10–15 cm/sec10,25 This resistance starts increasing on day 23 of the cycle in a nonconception cycle to RI 0.5–0.55. Endometrial Doppler shows branches of spiral arteries piercing the with RI 0.48–0.52. Uterine artery at this time of the cycle has PI of 2.0–2.5 and PSV 15–20 cm/sec (Figs 7.12A and B).
Based on these values abnormal parameters indicate luteal phase problems like luteinized unruptured follicle or luteal phase defect.
 
Luteinized Unruptured Follicle
On 2D US shows persistent follicle with thick walls and progressive loss of cystic appearance and is difficult to differentiate from corpus luteum. Endometrium is thick and echogenic and no fluid is seen in POD. On Doppler perifollicular RI is 0.51–0.59, which is higher than normal and remains almost normal till the end of the cycle. Nondominant ovary also shows similar Doppler indices. Endometrial flow is also absence.10
107
Figs 7.12A and B: Doppler scan in secretory phase: (A) Corpus luteum flow (B) Endometrial flow
 
Luteal Phase Defect
On Doppler corpus luteum shows high resistance flow with RI: 0.58 + 0.04. Dominant and nondominant ovaries show similar resistance indices. Increased resistance is also seen in spiral arteries RI: 0.72 + 0.06.26
108
Segmental uterine and ovarian artery perfusion demonstrate a significant correlation with histological and hormonal markers of uterine receptivity and may aid assessment of luteal phase defect.
Golan et al Shoham et al, and Tan SL et al quoted in this regard as follows:
“In the hands of experienced operators, ultrasound alone suffices for cycle monitoring, with no necessity for additional hormonal estimations.”
REFERENCES
  1. KupesicS, KurjakA. Predictors of in vitro fertilization outcome by three dimensional ultrasound. Hum Reprod 2002; 17(4): 950–55.
  1. Elien RKlinkert, Fertil Steril 2005; 83: 811–4.
  1. Mei-JouChen et al. Hum Reprod, 23 (4): 952–957; Feb. 2008.
  1. Deb,S; Jayaprakasan,K; Campbell,BK; Clewes,JS; Johnson,IR; Raine-Fenning,NJ Ultrasound in Obstetrics and Gynecology, Volume 33, Number 4, April 2009, p. 477–483 (7)
  1. TanSL. Clinical Applications of Doppler and three dimensional ultrasound in assisted reproductive technology. Ultrasound Obstet Gynecol 1999; 13: 153–6.
  1. BassilS, MagritteJP, RothJ, NisolleM, DonnezJ, GordtsS. Uterine vascularity during stimulation and its correlation with implantation in in vitro fertilization. Hum Reprod 1995; 10: 1497–501.

  1. 109 BourneT, JurkovicD, WaterstoneJ, CampbellS, CollinsWP. Intrafollicular blood flow during human ovulation. Ultrasound Obstet Gynecol 1991; 1: 53–9.
  1. KupesicS, KurjakA Ulterine and ovarian perfusion during the periovulatory period assessed by transvaginal colour dopler. Fertil Steril 1993; 3: 439–43.
  1. NagoriCB, PanchalSY. Double IUI in cases with high pre hCG perifollicular PSV for better pregnancy rates: a randomized study of 350 IUI cycles. Presented at 16th World Congress on Ultrasound in Obstetrics and Gynecology, 2006, London. J Ultrasound Obstet Gynecol Sept. 2006; 28(4): 612.
  1. KupesicS, KurjakA. The assessment of normal and abnormal luteal function by transvaginal colour Doppler sonography. Eur J Obstet Gynecol 1997; 72: 83–7.
  1. Kyei-MensahA, ZaidiJ, PittrofR, ShakerA, CampbellS, TanSL. Transvaginal three dimensional ultrasound: Accuracy of follicular volume measurements. Fertil Steril 1996; 65: 371–6.
  1. FeichtingerW. Transvaginal three dimensional imaging for evaluation and treatment of infertility. In MerzE (Ed): 3D Ultrasound in Obstetrics and Gynecology. Lippincott Williams and Wilkins  Philadelphia: 1998; 37–43.
  1. PanchalSY, NagoriCB. Can 3D PD be a better tool for assessing the pre hCG follicle and endometrium? A randomized study of 500 cases. Presented at 16th World Congress on Ultrasound in Obstetrics and Gynecology, 2006, London. J Ultrasound Obstet Gynecol. Sept. 2006; 28(4): 504.
  1. GonenY, CaspersRF. Prediction of implantation by the sonographic appearance of the endometrium during controlled ovarian stimulation for IVF. J In Vitro Fertil Embryo Transfer 1990; 7: 46.
  1. ApplebaumM. The ‘steel’ and ‘teflon’ endometrium-ultrasound visualization of endometrial vascularity in IVF patients and outcome. Presented at The third World Congress of Ultrasound in Obstetrics and Gynecology. Ultrasound Obstet Gynecol 1993; 3 (Suppl 2): 10.
  1. ZaidiJ, CampbellS, PittrofR, TanSL. Endometrial thickness, morphology, vascular penetration and velocimetry in predicting implantation in an in vitro fertilization program. Ultrasound Obstet Gynecol 1995;6: 191–8.

  1. 110 CheinLW, et al. Assessment of uterine receptivity by the endometrial-subendometrial blood flow distribution pattern in women undergoing IVF-ET. Fertil Steril 2002; 78: 245–51.
  1. CoulamCB, SternJJ, SoenksenDM, BrittenS, BustilloM. Comparison of Pulsatility indexes on the day of oocyte retrieval and embryo transfer. Hum Reprod 1995; 10: 82–4.
  1. Luis TMerce et al. 2D and 3D power doppler Ultrasound study of endometrium as implantation marker, textbook of transvaginal sonography 241–242.
  1. RagaF, et al., Assessment of endometrial volume by three dimensional ultrasound prior to embryo transfer: Clues to endometrial receptivity. Hum Reprod 1999; 14; 2851–4.
  1. KupesicS et al. Assessment of endometrial receptivity by transvaginal colour Doppler and three dimensional power Doppler ultrasonography in patients undergoing invitrofertilization procedures. J. Ultrasound Med 2001; 20: 125–34.
  1. WuHM, et al. Detection of subendometrial vascularization blood flow by three dimensional ultrasound may be useful for predicting pregnancy rate for patients undergoing in vitro fertilization-embryo transfer. Fertil Steril 2003; 79: 507–11.
  1. BaldR, HackeloerBJ. Ultraschall-darstellung verschiendener Endometrium for men. In OttoR. JanFX eds. Ultraschalldiagnostik 1982. Thieme.  Stuttgart: 1983: 187.
  1. GlockJL, et al. Fertil Steril 1995, 64: 500–4.
  1. SalimA, KurjakA, ZaludI. Ovarian Luteal flow in normal and abnormal early pregnancies. J Matern Fetal Invest 1992; 2: 119.
  1. KupesicS, KurjakA, VujisicS, PetrovicZ. Luteal phase defect: comparison between Doppler velocimetry, histological and hormonal markers. Ultrasound Obstet Gynecol 1997;9: 105–12.

Semen Preparation Techniques for IUI8

112Ejaculated semen is a mixture of spermatozoa and seminal plasma, which is constituted by secretions of epididymis, seminal vesicle and prostate. It may also contain other cellular components like microorganisms and leukocytes. The gist of different semen preparation techniques is to separate, the most functional spermatozoa from this ejaculate with minimal damage to sperms.
 
ADVANTAGES OF SEMEN PREPARATION
Removal of seminal plasma during semen preparation has other benefits also like:
  1. Insemination with raw semen can lead to pelvic infection as it may be contaminated with bacteria and other microorganisms.
  2. Seminal plasma is a rich source of prostaglandins that can lead to uterine contractions if inseminated in high concentration.
  3. Sperm washing significantly reduces the viral load. It is an ideal treatment option when the male partner is HIV positive.
  4. Antibodies in seminal plasma are removed with semen preparation. It also removes antigenic proteins, which may stimulate an immune reaction in females.
  5. It helps in removing decapacitation factors and other detrimental factors like dead cells and debris.
  6. Besides, during semen preparation certain active substances are added which improve the motility of spermatozoa.
113
 
TYPES OF TECHNIQUES
Over the years several techniques have been developed for semen preparation prior to IUI. The method used to prepare a semen sample depends on the following:
  1. Volume
  2. Viscosity
  3. Motile sperm count
  4. Presence of leukocytes, other cells and debris.
The most commonly used methods of semen preparation for IUI are:
  1. Methods based on self-migration of spermatozoa (i.e. swim up).
  2. Overlay method (swim up without centrifugation).
  3. Density gradient centrifugation (DGC) using different density gradient preparations.
Preparation methods, which do not use centrifugation, are in general advantageous. A normal ejaculate contains a large number of defective spermatozoa and granulocytes which on centrifugation can generate a high number of ROS (reactive oxygen species) and sperm DNA damage.13 These ROS in turn attack fatty acids present in plasma membrane of the sperm, resulting in oxidative stress which can damage sperm function.
 
Swim Up Technique (Figs 8.1A and B)
It is the earliest and most widely used technique of sperm preparation.4 In this procedure, the innate capacity of motile sperms to migrate against gravity is used to select a motile sperm population, with leaving behind seminal plasma, immotile sperms, extranuous cells and debris. It is a simple, easy to perform and a cost-effective technique.
114
Fig. 8.1A: Labelling of tubes
Fig. 8.1B: Mixing of 1 ml semen and 3–4 ml media
115Another advantage is that a good number of highly motile sperms are recovered. It is also effective for the samples which are collected in medium. It is restricted for normal or marginally abnormal ejaculates only as it does not separate morphologically normal sperms from abnormal ones (Fig. 8.2).
  1. Take 1 ml of semen sample in a conical tube.
  2. Add 1 ml of preincubated sperm preparation medium and mix.
  3. Centrifuge for 10 min at 1500 rpm (Balance centrifuge with a tube filled with same volume of water).
  4. Aspirate out supernatant without disturbing the pellet.
  5. Gently layer the pellet with 1 ml of medium.
  6. Keep the tube inclined in a CO2 incubator for 30–40 min.
  7. Determine the count and motility of supernatant.
  8. Carefully take the supernatant for insemination discarding the pellet (Figs 8.3 to 8.9).
 
Overlay Method
Basically, this technique is performed in the same way as standard swim up but avoids centrifugation which might damage spermatozoa. It is useful only for ejaculates with normal semen parameters that are samples with high degree of progressive motile spermatozoa. It cannot be used for viscous samples (Fig. 8.10).
  1. One ml of semen sample is overlaid with 1 ml of culture media.
    116
    Fig. 8.2: Swim up technique
    117
    Fig. 8.3: Placing the tubes in the centrifuge
    Fig. 8.4: Removal of supernatant
    118
    Fig. 8.5: Pellet
    Fig. 8.6: Overlaying of pellet with media
    119
    Fig. 8.7: Taking out the supernatant with motile sperms for insemination
    Fig. 8.8: Loading Makler to check sperm count
    120
    Fig. 8.9: Post wash sperm count
  2. Keep in incubator for 60–90 min at 37°C. During this time motile sperms migrate to culture medium.
  3. Aspirate out upper buffy layer for insemination (Figs 8.11 to 8.17).
 
Density Gradient Method
Basis of this method is that it separates motile spermatozoa from other components of seminal plasma based on their size, motility and specific density; sperms which are most progressively motile along the density gradient reach to the higher density portion at the bottom of the tube. Therefore, a sample devoid of dead sperms and debris and containing highly motile spermatozoa, with optimal morphology is attained.
121
Fig. 8.10: Direct overlay method
However, it is more expensive and time consuming and the lower recovery rate remains as an issue.
  1. Take 1 ml of 80% density gradient in a conical tube with a sterile pipette.
    122
    Fig. 8.11: Labeled semen sample
    Fig. 8.12: Semen sample
    123
    Fig. 8.13: Sample overlaying
    Fig. 8.14: Layered 0.5 ml semen with 0.5 ml media
    124
    Fig. 8.15: After swim up of sperms
    Fig. 8.16: Taking out supernatant with motile sperms for insemination
    125
    Fig. 8.17: After taking out supernatant
  2. Gently overlay it with 1 ml of 40% sperm gradient medium.
  3. Layer 1 ml of semen sample on the top of two layers.
  4. Without disturbing the layers, centrifuge it at 1500 rpm for 20 min (Fig. 8.18A).
  5. Discard the supernatant, leaving the pellet with as little of the 80% solution as possible.
  6. Take a new test tube and add 5–10 ml of medium. Transfer the pellet to this tube.
  7. Centrifuge it at 1500 rpm for 10 min (Fig. 8.18B).
  8. Remove and discard the supernatant without disturbing the pellet. This supernatant contains debris, leukocytes and the abnormal sperms.
  9. Resuspend the pellet in 1 ml of sperm wash medium. This is ready for insemination.
    126
    Fig. 8.18A: Density gradient method
    127
    Fig. 8.18B: Density gradient method—10 min centrifugation
    128
  10. Do post wash examination for sperm count and motility (Figs 8.19 to 8.35).
Fig. 8.19: Labeling of semen sample
Fig. 8.20: Checking for liquefaction
129
Fig. 8.21: Labeling of the conical tube
Fig. 8.22: Gradient media
130
Fig. 8.23: Layering 80% density gradient
Fig. 8.24: Layering 40% density gradient
131
Fig. 8.25: Overlaying of 80% media with 40% density gradient media
Fig. 8.26: Layering of density gradient with semen
132
Fig. 8.27: After layering of density gradient with semen
Fig. 8.28: Centrifuge with settings
133
Fig. 8.29: Gradient pellet
Fig. 8.30: Pellet drop
134
Fig. 8.31: Centrifuge with settings (Second centrifugation)
Fig. 8.32: Pellet after second centrifugation
135
Fig. 8.33: Reconstitution of pellet
Fig. 8.34: Loading Makler to check sperm count
136
Fig. 8.35: Post wash sperm
 
Other Techniques
 
Simple Wash
Sample is mixed with culture medium (3:1) and centrifuged. Supernatant is discarded, pellet resuspended and examined.
The simple wash method generally recovers the highest number of spermatozoa from a given ejaculate. It is useful in cases of severe oligospermia. However, the inseminate retains a mixture of motile, immotile, and immature sperm as well as nonspermatozoal cellular elements found in the semen.
137
 
Glass Wool Filtration
Here filtration is done through a glass wool column. It is not used routinely.
 
Sperm Select Method
It is a modification of overlay method where instead of culture medium, hyaluronic acid is used. The advantage of hyaluronic acid is that it mimics cervical mucus in composition5 but it requires spermatozoa of a fairly good motility.
 
COMPARISON OF VARIOUS METHODS
In a RCT in 2005, Soliman et al6 compared wash and pellet method with DGC. Wash and pellet method was found to be more cost-effective, took significantly less time and had same pregnancy rate as the gradient method. In another study of 166 couples, Nayar7 et al compared various semen preparation techniques for IUI and found that swim up has higher pregnancy rates and is more cost-effective than DG. Gradient method was found to be useful for poor quality samples. Simple washing is effective in samples with extremely low sperm densities. In another study Pranav8 et al found density gradient with percoll produces a significantly greater number of sperms with normal morphology than swim up method. In a recent Cochrane review9 various techniques were compared. They included 5 RCTs including 262 couples in total. There was no evidence of a difference between pregnancy rates for swim up versus a gradient or wash and centrifugation technique (OR 1.57, 95% CI 0.74 to 3.32; 138OR 0.41, 95% CI 0.15 to 1.10, respectively); nor in the two studies comparing a gradient technique versus wash and centrifugation (OR 1.76, 95% CI 0.57 to 5.44). There was insufficient evidence to recommend one technique over the other.
 
PROBLEM SAMPLES
 
Viscous Samples
A sample which fails to liquefy within 30 min requires liquefaction before processing. Passing the sample through a 23-gauge needle is usually effective.
 
Excessive Pus Cells
In general these samples should be discarded and a semen culture should be done to rule out any infection. If culture is sterile, repeated washing should be done to reduce the load of infection.
 
Low Volume
Sometimes, there is only a drop of semen. This drop should be immediately dissolved in medium to prevent any drying and then processed normally. For future, sample should be collected in medium.
 
Teratozoospermia
Density gradient technique is better in these cases.
 
Antisperm Antibodies
Sample should be collected in medium to avoid tagging antibodies to spermatozoa. It significantly reduces the percentage of antibody bound sperm.10
139
 
HIV Infection
There is a growing number of serodiscordant couples where only one partner is infected. Semen does contain virus particles and it has been seen that sperm washing can significantly reduce the viral load.11,12
REFERENCES
  1. AitkenRJ, ClarksonJS. Significance of reactive oxygen species and antioxidants, in defining the efficacy of sperm preparation techniques. J Androl 1988;9: 367–76.
  1. MortimerD. Sperm preparation techniques and iatrogenic failures of in-vitro fertilization. Hum Reprod 1991; 6: 173–6.
  1. ZiniA, MakV, PhangD, JarviK. Potential adverse effect of semen processing on human sperm deoxyribonucleic acid integrity. Fertil Steril 1999; 72: 496–9.
  1. DreviusLO. The ‘sperm-rise’ test. J Reprod Fertil 1971; 24: 427–9.
  1. ZimmermanER, RobertsonKR, KimH, DrobnisEZ, NakajimaST. Semen preparation with the Sperm Select System versus a washing technique. Fertil Steril 1994; 61: 269–75.
  1. SolimanS, GoyalA, RCT comparing two different methods of sperm preparation Fertil Steril 2005; 84, Supplement 1, S156.
  1. NayarKD, SehgalP, TiwariA. P-994: Comparative study of various semen preparation techniques in IUI and their effect on pregnancy rate Fertil Steril 2006; 86, S502–3.
  1. PranavPrakash, LucyLeykin, et al. Preparation by differential gradient centrifugation is better than swim up in selecting sperm with normal morphology (strict criteria). Fertil Steril 1998; 69.
  1. Cochrane Database Syst Rev. 2007, 17;(4):CD004507.
  1. Byrd, et al. Treatment of antibody associated sperm with media containing high serum content: a prospective trial of fertility involving men with high antisperm antibodies following intrauterine insemination. Am J Reprod Immunol 1994; 31: 84–90.

  1. 140 Savasi, et al. Safety of sperm washing and ART outcome in 741 HIV-1 discordant couples. Hum Reprod 2007; 22: 772–7.
  1. MencagliaL, FalconP, et al. ICSI for treatment of human immunodeficiency virus and hepatitis virus –serodiscordant couples with infected male partner. Hum Reprod 2005; 20: 2242–6.

Technique of Intra­uterine Insemination9

142Artificial insemination involves the deposition of semen in the vagina, cervix or uterine cavity, by means other than sexual intercourse (Table 9.1).
 
INTRAUTERINE INSEMINATION (IUI)
The IUI is the most commonly used method of insemination. It has six basic steps (Table 9.2).
143
 
TIMING OF INSEMINATION
The main objective is to time the insemination as close to the ovulation as possible. Four hours before or within 12 hours after ovulation is good enough to yield pregnancy. The rationale behind this timing is that viable spermatozoa should be present in the female genital tract at the time of ovulation. It is especially important in cases where cryopreserved sperms are used, as they do not have the longevity of fresh sperms.
The two most widely used methods to determine the time of ovulation are home monitoring of urinary LH for a spontaneous LH surge and timing of human chorionic gonadotropin (hCG) administration to trigger ovulation on the basis of ultrasound parameters. Since the actual moment of ovulation is not known, knowledge of the time course relationship between the LH surge or administration of hCG and ovulation is important.
 
DETECTION OF LH SURGE
LH surge itself is not a direct marker of follicular rupture, but it is an event which takes place approximately 24–36 hours before ovulation. Luteinizing hormone acts as a major paracrine regulator, and when a surge occurs, it triggers the final oocyte maturation (meiosis).
LH surge can be detected using ovulation predictor kits, which show a positive result when the urinary concentration of LH exceeds the threshold level (>40 mIU/ml for ELISA kit). This level is seen normally only during the period of LH surge.
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The test is done once daily, starting from the day the follicle approaches sonographic maturity. An IUI is done 24 hours after the LH surge test turns positive. It provides a cost-effective and patient-friendly way of recording follicular development and ovulation.
Disadvantages of this method are:
  • Results may be false-negative when the peak LH concentrations are low (<40 mIU/ml). False-negatives may occur in up to 25% of cycles and false positives are seen in up to 4% of cycles.13
  • Many ovulatory women have cycles in which the duration of the LH surge is too short to be easily detected or the concentration of LH is below the limit of detection because of hydration4, and hence dilution in the urine.
  • Women with ovulatory dysfunction might be less likely to experience or detect a LH surge.
  • The exact time at which ovulation occurs after the LH surge begins cannot be known earlier. It varies from 24 to 56 hours. Oocyte-fertilization capacity and sperm lifetime are <1 day and 1.4 days, respectively.5 Insemination needs to be performed close to ovulation time, and accurate synchronization is compulsory.
 
OVULATION AFTER hCG INJECTION
Follicular rupture takes place 36–48 hours after receiving the hCG injection. It offers the advantage of precisely timing the insemination, which is done 36 to 40 hours after hCG injection. An ultrasound examination is carried 145out at the time of or after the insemination. If there is no rupture of the follicle, an additional insemination is carried out the next day, 24 hours later.
Administration of hCG for IUI timing, has been accepted as the standard of care, for patients who are undergoing insemination for various infertility causes. A well-proven biological base, clinical predictability and ease of planning are some of the major advantages of this method. Disadvantages include the cost of the medication and the time-consuming ultrasonographic follicular monitoring, which add to the indirect costs of the method. Secondly, there is the theoretical risk of an increase in the rate of release of immature oocytes or of luteinized unruptured follicles.6,7
Several studies have been done comparing human chorionic gonadotropin (hCG) administration vs luteinizing hormone (LH) monitoring for intrauterine insemination timing. A meta-analysis5 done in 2007, found LH recording more useful than hCG administration in the male infertility group. The same association was observed in the unexplained infertility group. However, in patients with ovulatory dysfunction, hCG administration achieved better pregnancy rates than LH recording. Importantly, in all of the three different infertility groups, results were not statistically significant.
Martinez et al published a prospective, randomized trial comparing urinary LH surge, to hCG timing, based on ovarian follicle size.8 The group using the urinary LH surge had a 20% per cycle pregnancy rate, whereas the 146rate for the hCG group was 9%, a difference that was not statistically significant. In another prospective randomized study, Zreik et al compared IUI timing methods, in clomiphene citrate-treated women. They also found no difference between the pregnancy rates4 in the two groups.
 
NUMBER OF INSEMINATIONS
Usually, the cervix acts as a reservoir of sperms, where they are stored in glands and mucus. This maintains a continuous presence of sperms in the fallopian tubes available for fertilization, whenever the egg enters the tube. To maintain this phenomenon, double insemination was suggested, one 24 hours after the hCG injection and the second one, 36–40 hours later. However, the efficacy of two versus one insemination, has not yet been proved. Numerous studies have shown no difference in outcome.9 It is seen that, if semen parameters are poor, the second semen sample may not be of good quality and a well-timed single insemination gives better pregnancy results. Therefore, it is better to avoid double insemination, in patients with male factor infertility.
 
SEMEN COLLECTION AND SAMPLE ASSESSMENT
  1. The container in which it is collected should be sterile and non-toxic.
  2. The sample should be collected in the IVF clinic premises, if possible.
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  3. The volume should be noted. Unusual color or odor may indicate infection.
  4. The semen container is placed in an incubator at 37ºC. The sperm sample is allowed to liquefy for 20 minutes. Sometimes, the semen fails to liquefy. In that case, one can take a 23-gauge needle attached to a 10 ml disposable syringe. The viscous semen is passed repeatedly through the needle to achieve liquefaction.
  5. A drop of liquefied semen is placed on the Makler chamber. This is examined under the standard or phase contrast binocular microscope. The number of sperms in ten small squares on the Makler chamber grid is counted. This number gives us the sperm count in million/ml. In case of severely oligospermic samples, the number of sperms in all 100 small squares is counted. The final count is then divided by 10. The resultant figure is the sperm count in million/ml.
  6. The number of motile sperms in the same squares is counted and the percentage of motility determined. It is important to determine the prewash motile sperm count, as it is a good predictor of successful outcome in IUI. A minimum count of 10 million/ml PWSC is a predictor of a good result. Also a prewash sample with forward progressive motility of >30% is considered good.
  7. One can also do a rapid staining of a drop of semen sample and perform morphological studies. This is done at a magnification of 200 × (10 × objective and 20 × eyepiece). The morphology can be classified, based on either the WHO or Tygerberg-Kruger strict criteria.
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SPERM PREPARATION
Please find detailed discussion of the same in the chapter on “Sperm Preparation” (Fig. 9.1)
 
Choice of Insemination Cannula
Many types of cannula are available. The ideal insemination cannula should be
  • Easy to use
  • Disposable
  • Non-toxic to gametes
  • Soft or semi rigid—they must be firm enough to negotiate the sometimes unrelenting curvature of the cervix, but soft enough to avoid any trauma to the endocervix and/or endometrium
  • Inert to the endometrial lining.
Fig. 9.1: Flushing media for IUI
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Various types of catheters for use in artificial insemination, are commercially available. Catheters vary in length, caliber and location of the distal port (end- or side-dispersion systems). They also differ in the degree of firmness and rigidity. Different brands are available like Wallace, Makler, Gynetics, and Cook, etc (Figs 9.2 to 9.7).
 
Soft Artificial Insemination Catheters
  1. The Wallace artificial insemination catheter is a soft, flexible, double-lumen catheter, utilizing a co-axial system. The inner catheter is 18 cm long, with a rounded tip, and utilizes a bilateral side dispersion design to help prevent contamination and blockage of the catheter during insertion (Fig. 9.5 and 9.7).
  2. The Cook Soft-pass artificial insemination catheter is also a soft, flexible, double-lumen, co-axial catheter system. The inner catheter is 19 cm long with a rounded tip. Its outer guide catheter is similar to the outer sheath of the Wallace artificial insemination catheter.
  3. The Gynetics artificial insemination catheter is also a soft, flexible, double-lumen, co-axial catheter system. It is 20.6 cm in length with a rounded tip. In addition, similar to the Wallace artificial insemination catheter, it uses two lateral ports at the distal end for intrauterine sperm distribution. It also comes with a solid, thicker outer catheter for bypassing the difficult cervix.
 
Firm Artificial Insemination Catheters
  1. The Makler IUI cannula and insemination device (Fig. 9.4) is composed of two main parts: (i) a cannula that can be fitted to the tip of a 1.0 ml tuberculin syringe and (ii) a carrier to hold the syringe.
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    Fig. 9.2: IUI catheter
    Fig. 9.3: Tomcat IUI catheter
    Fig. 9.4: Makler insemination device
    The Makler IUI catheter (Fig. 9.6) is a firm, semi-rigid, single-lumen catheter with a rounded tip. It has a flared-base shape, which serves to seal the cervix at the time of insemination to help prevent the backflow of the injected contents.
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    Fig. 9.5: Wallace catheter
    Fig. 9.6: Makler catheter
    Fig. 9.7: Flushing the IUI cannula
    152
    This is accomplished by entirely obliterating the cervical external os by the flared base of the cannula. The clamp is then fixed to the outer brim of the speculum and the tension of the spring is adjusted so that the cannula is gently pressed against the cervical outlet.
  2. The Tomcat artificial insemination catheter (Fig. 9.3) is a firm, semi-rigid, single-lumen catheter. It is 11.4 cm long, 3.5 French (Fr) and with an open-end design. Originally, it was designed for draining the bladders of male cats, hence its name. It is designed with a semi-rigid but flexible body which, combined with its inherent memory, enables the catheter to be moulded to the curvature of the uterus.
Several studies have compared different catheters for IUI but no one catheter has been found to be superior to the other.1013 A meta-analysis14 done in 2006 compared the use of soft versus firm catheters in subfertile women undergoing IUI. No statistically significant difference was found in the clinical pregnancy and live birth rates per woman. As for the secondary outcomes, multiple pregnancy rates per clinical pregnancy were also not significantly different. More procedure-related difficulty was noted with soft catheters and more patient discomfort with firm catheters. Bleeding following the procedure was similar between the two groups.
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LOADING THE IUI CATHETER (Fig. 9.8)
  1. Check the label of the prepared sperm container and confirm the name with the patient.
  2. Flush the IUI cannula with 1–2 ml of flushing media. This will help to wash away any toxic factors present.
  3. Attach the cannula to the tuberculin syringe.
  4. Aspirate the desired quantity by immersing the tip in the prepared sperm suspension.
  5. Avoid contact of the cannula, with the wall of the test tube.
Fig. 9.8: Loading of semen sample in IUI catheter
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VOLUME OF INSEMINATION
In the uterine cavity, two layers are touching each other. While smaller volumes may not reach the fallopian tubes, a larger volume may cause distension of the uterine cavity, causing pain, discomfort, and backflow. Since fertilization occurs in the fallopian tubes, it is important to ensure that sperms reach there. It has been seen that an insemination volume of more than 0.4 ml reaches the fallopian tubes.15 Normally an insemination volume of 0.4 ml to 0.6 ml is used. A minimum concentration of 1 million motile sperms is essential. Good results are seen with an inseminate having more than 5 million motile sperms.
 
PROCEDURE FOR IUI
 
Prerequisites
  • Check consent.
  • Check label and data record of specimen being used.
  • Count and motility of processed sample is assessed.
  • Sample should be kept at 37ºC in the CO2 incubator.
  • The room where the procedure is done should be near the laboratory (Fig. 9.9).
 
Method
  • Patient is asked not to pass urine for 1–2 hours before IUI.
  • She is given the lithotomy or dorsal position.
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    Fig. 9.9: Instrument trolley for IUI
  • Sterile plastic gloves are used instead of latex gloves, as the powder of the latex gloves can be toxic to the sperms.
  • The perineum is cleaned with normal saline and the patient is draped.
  • The cervix is visualized using a Cuscos bivalve speculum or sims speculum in combination with an anterior vaginal wall retractor.
  • The vagina is prepared with normal saline. Avoid using any antiseptics, as it will kill the sperm.
  • Gently introduce the cannula into the uterine cavity through the cervical canal. Normally it is easy to pass a cannula into the uterine cavity. In difficult inseminations, where it is difficult to negotiate the canal, one can try to use a more rigid cannula.
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  • Deposit the inseminate very slowly into the uterine cavity over a three-minute period. After the deposit, the cannula is withdrawn very slowly. This prevents the sudden gushing out of inseminate which may happen if cannula is suddenly withdrawn.
  • Once the IUI is completed, the speculum is removed, and a head low position is given to the patient, who is then kept lying on the table for 15 minutes (Figs 9.10 to 9.12).
Fig. 9.10: Preparing the patient before IUI
157
Fig. 9.11: Exposing the cervix
Fig. 9.12: Insertion of IUI cannula through the cervix
158
 
Post IUI Instructions
  • Ask the patient to inform if she experiences abdominal pain, cramps or fever.
  • Start progesterone support.
  • Do a pregnancy test after 14 days.
  • Couple can have intercourse at any point after an IUI is performed.
  • She can return to work if she wishes to.
REFERENCES
  1. MillerPB, SoulesMR. The usefulness of a urinary LH kit for ovulation prediction during menstrual cycles of normal women. Obstet Gynecol 1996; 87: 13–7.
  1. NielsenMS, BartonSD, HatasakaHH, StanfordJB. Comparison of several one-step home urinary luteinizing hormone detection test kits to OvuQuick. Fertil Steril 2001; 76: 384–7.
  1. GuermandiE, VegettiW, BianchiMM, et al. Reliability of ovulation tests in infertile women. Obstet Gynecol 2001; 97: 92–6.
  1. ZreikTG, Garcia-VelascoJA, HabbooshMS, et al. Prospective, randomized, crossover study to evaluate the benefit of human chorionic gonadotropin-timed versus urinary luteinizing hormone-timed intrauterine inseminations in clomiphene citrate-stimulated treatment cycles. Fertil Steril 1999; 71: 1070–4.
  1. Ioannis P.Kosmas, AthinaTatsioni, HumanMusavi Fatemi, et al. Human chorionic gonadotropin administration vs. luteinizing monitoring for intrauterine insemination timing, after administration of clomiphene citrate: a meta-analysis Fertil Steril 2007; 87 607–12.
  1. CheckJH, PeymerM, ZaccardoM. Evaluation of whether using hCG to stimulate oocyte release helps or decreases pregnancy rates following intrauterine insemination. Gynecol Obstet Invest 1994; 38: 57–9.

  1. 159 CoetsierT, DhontM. Complete and partial luteinized unruptured follicle syndrome after ovarian stimulation with clomiphene citrate/human menopausal gonadotropin/human chorionic gonadotropin. Hum Reprod 1996; 11: 583–7.
  1. MartinezAR, BernadusRE, VoorhorstFJ, et al. A controlled study of human chorionic gonadotropin induced ovulation versus urinary luteinizing hormone surge for timing of intrauterine insemination. Hum Reprod 1991; 6: 1247–51.
  1. CantineauAEP, HeinmanMJ et al. Single versus double intrauterine insemination in stimulated cycles for sub fertile couples. Cochrane Database of Systematic reviews 2003, Cd003854.
  1. SegalS, TaoX, ShifrenJ, et al. The effect of insemination catheter type on the outcome of intrauterine insemination cycles. Fertil Steril 1998; 70: S146.
  1. SmithKL, GrowDR, WiczykHP, et al. Does catheter type effect pregnancy rate in intrauterine insemination cycles? J Assist Reprod Genet 2002; 19: 49–52.
  1. MillerPB, AcresML, ProctorJG, et al Flexible versus rigid intrauterine insemination catheters: a prospective, randomized, controlled study. Fertil Steril 2005; 83: 1544–6.
  1. FancsovitsP, TothL, MurberA, et al. Catheter type does not affect the outcome of intrauterine insemination treatment: a prospective randomized study. Fertil Steril 2005; 83: 699–704.
  1. Ahmed M.Abou-Setta, Ragaa T.Mansour, Hesham G.Al-Inany, et al. Intrauterine insemination catheters for assisted reproduction: a systematic review and meta-analysis. Hum Reprod 2006 21: 1961–7.
  1. FrancoJG, BaruffiRLR, MauriAL, et al. Radiological evaluation of incremental intrauterine instillation of contrast material. Fertil Steril 1992; 58: 1065–7.

Luteal Phase Support in IUI10

162Following ovulation, the luteal phase of natural cycle is characterized by the formation of corpus luteum, which secrets steroid hormones including progesterone. Normal luteal function is essential for maintenance of pregnancy. Several studies have shown that removal of corpus luteum during early pregnancy results in complete abortion.1,2
A meta-analysis of randomized traial indicated that luteal phase support led to significantly higher pregnancy rates than placebo in ART cycles. In another randomized study,4 pregnancy rate after IUI was found to be significantly high in patients with luteal support. These data suggest that a good luteal phase support is necessary for the maintenance of the early pregnancy and positively affects the success of COH and IUI cycles.
 
WHY LUTEAL SUPPORT IS NEEDED IN CONTROLLED OVARIAN STIMULATION WITH IUI?
Luteal support is needed after IUI because.
  • Excessively high estrogen levels seen in controlled ovarian stimulation protocols may induce premature luteolysis.
  • Some protocols may give only pure FSH, thus leading to relatively low LH values.
  • Some protocols use GnRH antagonist to prevent premature LH surge or to avoid IUI on weekends. Though they are administered for a short period and have a short duration of effect, their impact on corpus luteum is still not known.
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  • Luteal phase support is given to counteract luteal insufficiency if present.
Initially hCG or progesterone was used to provide luteal phase support. Over the years, however, progesterone has become the drug of choice because hCG was found to be associated with a higher risk of ovarian hyperstimulation syndrome.58 Most treatment protocols advocate the use of progesterone throughout the first trimester of pregnancy, based on the findings of Shamma et al9 who used 17-hydroxyprogesterone as a marker to demonstrate ongoing corpus luteum activity up to 10 weeks of pregnancy.
 
TYPES OF PROGESTERONE
 
Natural Progesterone (Micronized Progesterone)
Various formulations of progesterone are now available including oral, vaginal, intramuscular (IM) and rectal.
Progesterone administered orally is subjected to significant prehepatic and hepatic first pass metabolism resulting into its degradation to various metabolites and decreased bioavailability. In addition to erratic absorption, plasma levels also are poorly sustained and returns to baseline within six hours. Morever, the metabolites formed act centrally and can cause sedation, headache and drowsiness. Patient may complain of urinary frequency and constipation also.
There is increased bioavailability and reduced variability when progesterone is administered vaginally. Plasma level remains elevated for up to 48 hours. This sustained level produces a more physiological endometrial 164response. It has a unique “uterine first pass’ effect where the uterine tissue has a higher than expected progesterone level, despite a lower serum progesterone.10 Furthermore, by bypassing prehepatic and hepatic “First pass metabolism” progesterone is not metabolized to byproducts that can cause dizziness and somnolence.
Several studies have compared oral vs.vaginal progesterone. Levine and Watson11 compared the pharmacokinetics of oral micronized progesterone (100 mg) with that of vaginal progesterone gel (crinone 8%, 90 mg). Results showed that vaginal gel was associated with a higher maximum concentration of progesterone. Furthermore, the 24 hours “Area under the Curve” for drug concentration vs time was higher in the group who had received vaginal progesterone. They concluded that the vaginal administration of progesterone results in a greater bioavailability with less relative variability than oral progesterone.
In another randomized study,12 investigators compared vaginal progesterone gel (crinone 8%) 90 mg once daily with an oral progesterone preparation uterogestan 400 mg once daily and Im progesterone in oil 50 mg once daily. The clinical and ongoing pregnancy rates were comparable between the vaginal gel and im groups, but significantly lower with the oral formulation.
Intramuscular route is the most reliable route to achieve the desired concentration of progesterone. However, it has the disadvantage of daily injections and pain and abscess formation at injection site. It has a rare risk of developing severe allergic reactions, ARDS and eosinophilic pneumonitis. Oil used in the preparation of 165intramuscular progesterone is derived from sesame oil. In case of known allergies, it is recommended to switch over to peanut oil preparations.
Cicinelli et al13 recently compared vaginal progesterone gel administration with Im injection. They found that the ratio of endometrial to serum progesterone concentrations was markedly higher in patients who had received vaginal progesterone gel. In another study,14 histological changes on endometrium were same after vaginal and Im administration of progesterone. This occurred despite the fact that serum levels were lower after vaginal gel administration than after Im administration.
All these studies strongly suggest that both vaginal and Im progesterone are effective, but vaginal administration is much easier and convenient for patients whereas orally administered progesterone appears to be inferior.
Vaginal route is the most preferred route of administration.
 
Other Progestogens
 
Dydrogesterone
Dydrogesterone has a dual advantage as it acts as immunomodulator also and thus prevents rejection of paternal antigen by mother. Marked immunomodulatory effect of dydrogesterone in maintaining a T helper-2 cytokine balance means that it is a good choice for preventing abortion in women suffering from subchorionic hemorrhage.
Exposure to dydrogesterone resulted in a significant inhibition in the production of the proinflammatory cytokines IFN-gamma and TNF-alpha and a significant 166increase in the levels of the anti-inflammatory cytokine IL-4, resulting in a substantial shift in the ratio of Th1/Th2 cytokines. It also induces a shift in cytokine bias, by inhibiting proinflammatory cytokine production and increasing anti-inflammatory cytokine production.
Dydrogesterone shares similar biological properties with natural progesterone and has high affinity for progesterone receptors. It is safe, well tolerated, non-androgenic and less in side effects. Dydrogesterone does not change the basal body temperature and ovulation can still be detected. Profile to endogenous progesterone, dydrogesterone finds specific relevance due to it being orally active at much lower doses. Its freedom from estrogenic, androgenic, anabolic, corticoid and other undesirable hormonal effects gives it additional benefits over most other progestins.
Doses of progesterone supplementation for early pregnancy support:
  1. Micronized progesterone 200–300 mg/day, orally or vaginally
  2. Injection progesterone 25–50 mg IM everyday or
  3. Oral dydrogesterone 20–30 mg/day.
 
DURATION OF SUPPORT
Support is started from the day of IUI. It is given initially for 14 days. At the end of 14 days, serum β-hCG is done for detection of pregnancy. If patient is pregnant, luteal support is continued until 10 to 12 weeks when placenta takes over the role of progesterone production.
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ROLE OF ANTIBIOTICS
The vagina is an area of the body that is abundant with normal bacterial flora. Therefore, any procedure through the vagina may be considered to have added potential for resulting in post-procedure infection. Prophylactic antibiotics may play a role in the prevention of post-procedure transcervical intrauterine infections.
Many units like to give prophylactic antibiotic for 5–7 days after IUI. However, there have been no randomized controlled trials evaluating the usefulness of antibiotics for the prevention of infection after these procedures.15
 
Case
32-year-old female, married for 3 and a half years with secondary infertility since 2 years. She conceived naturally within a year of her marriage but had a spontaneous abortion at 6 weeks and now presented with secondary infertility. She had history of short cycles. Her blood investigations revealed a high serum prolactin levels for which she received Tab cabergoline 0.25 mg once a week. HSG showed a normal uterine cavity with bilateral free spillage of dye. Husband semen analysis revealed no abnormality. She was planned for intrauterine insemination. Ovulation induction was done using Clomiphene 168citrate 100 mg from day 2 to day 6. Recombinant FSH 75 U was administered on day 7, day 8 and day 9. Her follicular monitoring revealed a dominant follicle on day 12. Injection hCG 10,000 U was given and IUI was performed after 36 hours. Dydrogesterone 10 mg twice daily was given from the day of the IUI. Her β-hcg 14 days later showed a positive result. Luteal support was continued till 12 weeks of pregnancy. She had an uneventful antenatal period and delivered normally at term.
REFERENCES
  1. GibsonWE, TonerJP, et al. Experience with a novel progesterone preparation in a donor oocyte program. Fertil Steril 1998; 69: 96–101.
  1. FatemiHM, Popovic-TodorovicB, et al. An update of luteal phase support in stimulated IVF cycles. Hum Reprod Update 2007; 13: 581–90.
  1. SolimanS, DayaS, CollinsJ, HughesEG. The role of luteal phase support in infertility treatment (a meta-analysis of randomized trials). Fertil Steril 1994; 61: 1068–76.
  1. AtmacaS, ErdemM, et al. The impact of luteal phase support on pregnancy rates in intrauterine insemination cycles; a prospective randomized study. Fertil Steril 2007; 88: S163.
  1. HermanA, Ron-ElR, GolanA, et al. Pregnancy rate and ovarian hyperstimulation after luteal human chorionic gonadotropin in in-vitro fertilization stimulated with gonadotropin-releasing hormone analog and menotropins. Fertil Steril 1990; 53: 92–6.
  1. MochterMH, HogerzeilHV, MolBW. Progesterone alone versus progesterone combined with hCG as luteal support in GnRHa/HMG induced IVF cycles (a randomized trial). Hum Reprod 1996; 11: 1602–5.

  1. 169 McClureN, LeyaJ, RadwanskaE, et al. Luteal phase support and severe ovarian hyperstimulation syndrome. Hum Reprod 1992; 7: 758–64.
  1. MacDougallMJ, TanSL, JacobsHS. In vitro fertilization and the ovarian hyperstimulation syndrome. Hum Reprod 1992; 7: 597–600.
  1. ShammaFN, PenziasAS, ThatcherS, et al. Corpus luteum function in successful in vitro fertilization cycles. Fertil Steril 1992; 57: 1107–9.
  1. BullettiC, de ZieglerD, et al. Targeted drug delivery in gynecology: the first uterine pass effect. Hum Reprod 1997; 12: 1073–9.
  1. LevineH, WatsonN. Comparison of the pharmacokinetics of Crinone 8% administered vaginally versus prometrium administered orally in postmenopausal women. Fertil Steril 2000; 73: 516–21.
  1. SaucedoLLE, GalacheVP, HernandezAS, et al. Randomized trial of three different forms of progesterone supplementation. Fertil Steril 2000, 74 (3S): S205.
  1. CicinelliE, DeZieglerD, BullettiC, et al. Direct transport of progesterone from vagina to uterus. Obstet Gynecol 2000; 95: 403–6.
  1. TavaniotouA, SmitzJ, BourgainC, DevroeyP. Comparison between different routes of progesterone administration as luteal phase support in infertility treatments. Hum Reprod Update. 2000; 6: 139–48.
  1. ThinkhamropJ, LaopaiboonM, LumbiganonP. Prophylactic antibiotics for transcervical intrauterine procedures. Cochrane Database of Systematic Reviews 2007, Issue 3. Art. No. CD005637.

Difficult IUI11

172The IUI is one of the safest, least invasive and most cost-effective forms of assisted reproduction. The procedure is simple and very easy to perform. Sometimes, however, one can come across an IUI which is not as easy as is thought and it may be difficult to negotiate the cervical canal. Difficulties may arise in presence of:
  • Acutely anteverted uterus
  • Acutely retroverted uterus
  • Stenosed cervix
  • Abnormal cervical canal
  • Apprehensive patient.
 
ACUTELY ANTEVERTED/RETROVERTED UTERUS
In such a situation there may be difficulty in negotiating the cannula. Certain maneuvers can help in this:
  • Full urinary bladder can partially correct the anteversion.
  • Gentle traction on the anterior cervical lip can straighten the uterocervical axis.
  • Insemination can be performed under ultrasound guidance.
 
STENOSED CERVIX
In case of stenosed cervix:
  • Catheter which is soft, pliable and with small outer diameter should be used.
  • Sonographic guidance may be used.
  • Dilatation of the cervix is usually not preferred as it causes trauma. However, it can be used as the last resort if it is not possible to negotiate the os even under sonographic 173guidance with a small catheter. Dilatation should be as minimum just enough to allow the insemination cannula. The dilator should not be pushed high into the uterine cavity as can traumatize the endometrium. It can be performed on first or second day of the menstrual cycle if difficulty was encountered in the previous cycle.
 
ABNORMAL CERVICAL CANAL
A malleable cannula (designed by Wallace) which can be given a desired shape can be used in such a situation. Insemination can be performed under ultrasound guidance to see the shape of cervical canal.
 
APPREHENSIVE PATIENT
Apprehensive patient can cause difficulties as patient cooperation is important for a successful IUI procedure. Patient counseling about the simplicity and ease of the procedure can help allay the anxiety or a mild sedative can sometimes be used for the procedure.
 
ROLE OF SONOGRAPHY IN DIFFICULT SITUATIONS
It provides the knowledge of length and direction of uterocervical canal. Cannula can be passed under the guidance of ultrasound and necessary changes can be made in the direction of the cannula as required.
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REFERENCES
  1. KyungEui Park, Seung-YupKu, HoonKim, ByungChul Jee, ChangSuk Suh et al. The Use of Tenaculum During Intrauterine Insemination may not Affect the Pregnancy Outcome. Yonsei Med J 2010 November 1; 51(6): 949–53.
  1. Ahmed M.Abou-Setta, Ragaa T.Mansour, et al. Intrauterine insemination catheters for assisted reproduction: a systematic review and meta-analysis. Hum Reprod 2006; 21 (8): 1961–67.
  1. DuranHE, MorshediM, KrugerT, OehningerS. Intrauterine insemination: A systematic review on determinants of success. Hum Reprod Update 2002; 8: 373–84.

Complications of IUI12

176Though IUI is a safe procedure, rarely complications can occur which may be directly or indirectly related to the procedure. The treating physician should have a complete knowledge and understanding of these complications to minimize there incidence and also should be capable of treating them in case it occurs. The complications can be divided as:
  • Due to ovarian stimulation
  • Due to insemination procedure
  • Other complications.
 
COMPLICATIONS DUE TO OVARIAN STIMULATION
Intrauterine insemination is usually combined with cont­rolled ovarian stimulation as natural cycle IUI is rarely done rare. Controlled ovarian stimulation can be associated with certain complications which can be divided as immediate and delayed:
 
Immediate
  • OHSS
  • Multiple pregnancies.
 
Delayed
  • Risk of ovarian cancer.
 
Ovarian Hyperstimulation Syndrome (OHSS)
The OHSS is an iatrogenic and one of the most dreaded complications associated with ovarian stimulation. It is associated with a wide spectrum of clinical signs and symptoms, ovarian enlargement, fluid shift from 177intravascular to extravascular compartment and changes in biochemical parameters. It is usually seen after stimulation with gonadotropins, however, can rarely occur after clomiphene citrate use.
 
Incidence
The incidence of OHSS ranges from 3 to 23% and varies according to the ovulation induction protocols and the risk profile of population being studied.1 The incidence of mild, moderate and severe OHSS is:
Mild
8–23%
Moderate
0.005–7%
Severe
0.008–2%
 
Pathophysiology
The exact pathophysiology is still not elucidated. The major events happening are neovascularization and increased vascular permeability that leads to acute fluid shift from the intravascular to extravascular compartment. This shift occurs due to release of vasoactive substances from the ovary under the influence of hCG. These are renin-angiotensin, interleukins, nitric oxide and vascular endothelial growth factor (VEGF).2 It has recently been identified that increased sensitivity of the FSH receptor to hCG is responsible for the spontaneous OHSS occurring during pregnancy.3
 
Classification of OHSS
OHSS has been classified into various stages and grades according to the clinical symptoms, signs, ultrasonographic findings and laboratory parameters. The most commonly used is the Golan's classification which has 178divided it in three stages: mild, moderate and severe and 5 grades (Table 12.1).4 The severest grade of OHSS is further classified by Navot's as severe and critical life-threatening depending on clinical and biochemical findings to indicate when management in an intensive care setting should be considered (Table 12.2).5 The purpose of clinical staging is to guide patient management. In general, mild and moderate forms of OHSS can be treated expectantly on OPD basis.
179
Those with severe form should be admitted and those with critical OHSS are best managed in consultation with other specialist in intensive care setting.
 
Complications of OHSS
  1. Vascular complications: venous compression, immobility and state of hypercoagulability can cause deep vein thrombosis. Cerebrovascular complications can occur secondary to these thromboembolic events.
  2. Liver dysfunction
  3. Renal dysfunction
  4. Respiratory compromise
  5. Adnexal torsion, rupture or hemorrhage
180
Fig. 12.1: Ultrasound image of an hyperstimulated ovary
 
Prediction
Various factors have been identified to help define patients at high risk of ovarian hyperstimulation syndrome. All patients undergoing ovulation induction should be closely monitored with:
  1. Ultrasound follicular monitoring: All follicles including the smaller ones should be measured. The small (< 9 mm) and intermediate follicles are more dangerous as these continue to grow and produce estradiol following hCG administration (Fig. 12.1).6
    Patients with PCOS are more likely to develop OHSS than those without PCO as these have high sensitivity to gonadotropin stimulation due to large cohort of FSH sensitive small antral follicles.7
    181
  2. Serum estradiol levels: A high level and a steep rise can predict likelihood of developing OHSS (Table 12.3).
 
Prevention
  1. Withholding hCG: hCG trigger is usually given when the follicles reach 16–25 mm in size and estradiol level reaches 200–400 pg/ml per leading follicle. hCG is withheld in an IUI stimulated cycle if serum estradiol levels are more than 1500 pg/ml. USG findings of more than 6 leading follicles can also be used a criteria to withhold hCG.
    Also, if estradiol levels are more than doubling during 2–3 days (steep slope) then it should be regarded as a serious warning sign and hCG should be withheld in that cycle.
    When hCG is withheld:
    • Follicles can be aspirated and embryos cryopreserved, provided GnRH analogs have been given.
      182
    • Continue GnRH agonists after stopping gonadotropins. Once downregulation occurs cycle can be restarted at a lower dosage.
  2. Delaying hCG (coasting): When estradiol levels are high hCG is withheld, GnRH agonist is continued and gonadotropins are stopped till E2 falls following which hCG is given.8 Withholding hCG causes apoptosis of granulosa cells and atresia of large number of follicles. Long coasting periods however have a negative impact on number of oocytes, implantation and pregnancy rates.9,10
  3. Decreasing dose of hCG: lower dose of hCG (5000 units instead of 10,000) may avoid hyperstimulation by exerting shorter periods of stimulation.
  4. Use of GnRH agonist instead of hCG for LH surge: As period of stimulation is lesser with GnRH agonist surge there is no hyperstimulation. The rates of fertilization, implantation, clinical pregnancy, ongoing pregnancy, and abortion rates observed are similar to hCG.11
  5. Follicle aspiration: Follicle aspiration can decrease the chance of OHSS.12 However, some studies contradict this.
  6. Albumin/hydroxyethyl starch: This increases the serum oncotic pressure and prevents leakage of fluid in the third space. This is used as a prophylactic measure. The disadvantage is that its oncotic action lasts only for 36 hours.
  7. Conversion to an IVF cycle, cryopreservation of embryos and subsequent transfer in a later cycle. This helps decreasing chances of OHSS due to pregnancy with the advantage of not loosing the cycle and replacing the frozen thawed embryos in a later cycle.
    183
  8. Steroids: Methyl prednisolone has been tried in cases of OHSS.13
  9. Cabergoline: This dopamine receptor agonist inactivates VEGF receptor and prevents increased vascular permeability. It is administered in a dose of 0.5 mg/d, starting from the day of hCG, for 8 days. The incidence of OHSS is significantly reduced.14
 
Treatment
The condition is self-limiting and usually resolves in 10–14 days. The treatment depends on the immaturity of the patient and tue severity of the disease (Table 12.4).
 
Mild OHSS
 
Grade I
The treatment is usually conservative and is done on an outpatient basis with a close follow-up. One should reassure the patient. She should be advised to have plenty of fluids and to avoid exertion.
 
Grade II
Minimize physical activity and take plenty of fluids. Analgesics and antiemetics may be used as required. Serum hematocrit and electrolytes should be monitored. Input-output record should be maintained. Reassessment is required if there is increase in weight more than 2 kg or if there is worsening of symptoms.
184
 
Indication of Hospitalization
  1. Grades II or III OHSS if:
    • Intolerable nausea or vomiting
    • Hypotension
    • Pleural effusion
    • Ascites
    • Hematocrit >48%
    • Sodium <135 mg/L
    • Potassium level >5.0 mg/L
    • Serum creatinine >1.2 mg
  2. All cases of grades IV or V
185
 
 
Aim
  • Correction of circulatory volume
  • Correction of electrolyte imbalance
  • Maintaining renal function
  • Prevention of thrombosis.
  1. Maintaining intravascular volume: As there is hypo­natremia normal saline with or without glucose is the main crystalloid used. Plasma expanders like albumin are also used as there is protein loss in the third space. Other fluids like mannitol, dextran and fresh frozen plasmas can also be used.
  2. Prevention of thrombosis: Low dose heparin can be used in cases where there is altered coagulation profile. Thromboembolic events require therapeutic anticoagulation with heparin.
  3. Diuretics: These are used if oliguria persist in spite of restoring intravascular volume or in case of pulmonary edema. If renal failure still does not resolve with these measures, dopamine is added to dilate the renal vasculature.
  4. Ascites: Paracentesis is done if there is severe discomfort; venous return is compromised, there is respiratory distress, renal compromise or hemoconcentration unresponsive to medical therapy. It should only be done if patient is hemodynamically stable.
  5. Paracentesis of hydrothorax is done in cases of pleural effusion leading to dyspnea. Severe respiratory compromise may require ventilatory support.
  6. Termination of pregnancy: If critical condition still does not improve, one may consider termination of pregnancy.
    186
  7. Laparotomy is required in cases of ovarian torsion, hemorrhage and rupture.
 
Multiple Pregnancies
This is an inevitable complication associated with ovarian stimulation. This is increased more when gonadotropins are used for ovarian stimulation compared to clomiphene citrate. It is very important to carefully monitor the patients undergoing COS plus IUI to minimize the incidence of multiple pregnancies. These births are associated with significant maternal morbidity and also preterm birth with its sequel of neonatal mortality and morbidity.
Women at high risk of multiple pregnancies are usually less than 30 years of age; with 6 or more than 6 preovulatory follicles and with peak serum estradiol more than 1000 pg/ml. The following options are available to such women:
  • Cancel the cycle and reinitiate with a lower gonadotropin dose in the next cycle.
  • Limited oocyte aspiration
  • Conversion to IVF
  • Proceed with the cycle and multifetal pregnancy reduction.
 
Risk of Ovarian Cancer
Earlier studies have suggested a three fold increase in incidence of ovarian cancer in women who undergo ovulation induction.15 However, the recent reports have not replicated these findings.16,17 Some reports however report an increased incidence of borderline tumors.18 It should be 187kept in mind that the cancers are over diagnosed in infertile women because of close medical surveillance.
It has however been recommended that these drugs should not be used consecutively for more than 6 cycles and not more than a total of 12 cycles. Smallest dose for shortest possible duration should be used.
 
COMPLICATIONS DUE TO THE PROCEDURE
These are rare and not life-threatening. These are:
  1. Infection
  2. Trauma and bleeding
  3. Pain
  4. Noninfective salpingitis
  5. Allergic reaction
  6. Antisperm antibody
  7. Vasomotor symptoms.
 
Infection
The risk of infection with IUI is rare and is estimated to be 1.8 per 1000 women.19 The reasons for such low risk are:
  • The population has already been screened for infection.
  • The risk of acquiring STD during treatment is rare.
  • Sperm preparation techniques are thought to remove microbes.20
Infection can however occur sometimes. The sources of infection are:
  • Local source: resident urethral flora, hands, glans penis
    188
  • Airborne bacteria in the semen collection room
  • Contamination due to faulty technique:
    • Collection in unsterile container
    • Nonsterile preparation techniques
    • Cannula tip touching the vagina
    • Contamination with cervical flora.
The organisms commonly isolated are E. coli, N. gonorrhoea, Trichomonas vaginalis, Streptococcus, Ureaplasma, etc.
 
Prevention
  • Aseptic techniques and quality control should be maintained.
  • Sperm wash media can be supplemented with antibiotics.
  • Educate patient on proper semen collection techniques.
 
Trauma and Bleeding
IUI is a relatively simple and easy procedure and proper technique can avoid trauma. Trauma and bleeding, however, can sometimes occur due to injury to the internal os or the endometrium by the insemination cannula or by injury to the cervical lip by Allis/Volsellum.
189
 
Pain
Pain can occur because of uterine cramps. Severe cramps have been reported in 6–17% of cases.22 Pain can be due to instrumentation. Also in cases of difficult IUI trauma to the endometrium can cause release of prostaglandins which can cause cramping pain. Prostaglandins can also be introduced with seminal plasma. Good semen washing techniques and reducing the volume of inseminate can minimize this problem.
 
Noninfective Salpingitis
Any foreign substance like sperm preparation ingredients like percoll can irritate the endometrium and can cause noninfective salpingitis. Proper washing of sperms after density gradients can prevent this.
 
Allergic Reaction
These are rare. Mild reactions may not be seen immediately but can occur later. Severe anaphylaxis can rarely occur. It can be due to semen itself, ingredients of sperm wash media or due to allergy to penicillin or bovine albumin.
 
Vasomotor Symptoms
Vasomotor symptoms such as nausea, bradycardia, and diaphoresis can occur rarely.
 
Antisperm Antibody
During IUI, a large dose of antigenic sperms is deposited into the uterine cavity directly, bypassing the cervix, 190which otherwise acts as a barrier and modifies the antigenic load. The sperms are then cleared by the macrophages into the peritoneal cavity. A high immunogenic load may increase the production of antibodies. Different women however have different immune response and most reports do not support the hypothesis of formation of antibodies with IUI.
 
OTHER COMPLICATIONS
  • Abortion
  • Ectopic pregnancy
  • Accidental insemination with wrong sample.
 
Abortion
Abortion rate with IUI is estimated to be 20–30%.23,24 This could be attributed to higher age of these women and increased incidence of multiple pregnancy. Early diagnosis and close monitoring of these patients compared to general population also results in higher reported abortion rates.
 
Ectopic Pregnancy
The incidence of ectopic pregnancy is higher in women undergoing IUI and other ART procedures. The incidence is higher in women with history of tubal disease. Even in those without such history the incidence is about 5 times higher than in general population.
191
 
Accidental Wrong Samples
When more than one sample is processed at one time, there is a possibility of mixing of samples. To avoid such a situation:
  • Label all samples
  • Use separate syringes and pipettes for separate samples
  • Properly identify the sample before loading the cannula.
  • More than one person should check the sample.
  • Gynecologist should recheck the identity before inseminating.
REFERENCES
  1. SchenkerIG, WeinsyeinD. Ovarian over stimulation syndrome: a current survey. Fertil Steril 1978; 30: 179–83.
  1. KosakaK, FujiwaraH, YoshiokaS, FujiiS. Vascular endothelial growth factor Production by circulating immune cells is increased in ovarian hyperstimulation syndrome. Hum Reprod 2007.; 22: 1647–51.
  1. De LeenerA, CaltabianoG, ErkanS, et al. Identification of the first germ line mutation in the extracellular domain of the follitropin receptor responsible for spontaneous hyper-stimulation syndrome. Hum Mutat 2007; 29: 91–8.
  1. GolanA, RonEl-R, HermanA, et al. Ovarian overstimulation syndrome: an update review. Obstet Gynecol Survey 1989; 44; 430–4.
  1. NavotD, BerghPA, LanferN. Ovarian overstimulation syndrome in novel reproductive technologies. Fertil Steril 1992; 58: 249–61.
  1. BlauksteinJ, ShalevJ, SaadonT, et al. Ovarian overstimulation syndrome: prediction by number and size of ovarian follicles. Fertil Steril 1987; 47: 597–602.

  1. 192 Van der MeerH, HompesPGA, de BoerJAM, et al. Cohort size rather than follicle-stimulating hormone thresholds level determines ovarian sensitivity in Polycystic ovarian syndrome. J Clin Endocrinol Metab 1998; 83: 423–26.
  1. MervielP, ClaeysC, HeraudH, et al. Coasting and ovarian stimulation protocols in high responder patients undergoing assisted conception. Gynecol Obstet Fertil 2005; 33: 703–12.
  1. OwjM, TehraniNejad ESh, AmirchaghmaghiE, et al. The effect of withholding gonadotropin (a coasting period) on the outcome of in vitro fertilization cycles. Eur J Obstet Gynecol Reprod Biol. 2007; 133: 81–5.
  1. CheemaP, GelbayaTA, HorneG, et al. The optimal length of ‘coasting protocol’ in women at risk of ovarian hyperstimulation syndrome undergoing in vitro fertilization. Hum Fertil 2006; 9: 175–80.
  1. ShapiroBS, DaneshmandST, GarnerFC, et al. Comparison of human chorionic gonadotropin and gonadotropin releasing hormone agonist foe final oocyte maturation in oocyte donor cycles. Fertil Steril 2007; 88: 237–9.
  1. ZhuWJ, LiXM, ChenXM, et al. Follicular aspiration during the selection phase prevents severe ovarian hyperstimulation in patients with polycystic ovarian syndrome who are undergoing in vitro fertilization. Eur J Obstet Gynecol Reprod Biol 2005; 122: 79–84.
  1. LainesT. et al. Administration of methyl prednisolone to prevent severe ovarian hyperstimulation in patients undergoing in vitro fertilization. Fertil steril 2002; 78: 529–34.
  1. AlvarezC, Marti-BonmatiL, Novella-MaestreE, et al. Dopamine agonist cabergoline reduces hemoconcentration and ascites in hyperstimulated women undergoing assisted reproduction. J Clin Endocrinol Metab 2007; 92: 2931–7.
  1. WhittemoreAS, HarrisR, ItnyreJ, et al. Characteristics related to ovarian cancer risk: collaboratie analysis of 12 US case controlled studies. I Methods. Collaborative ovarian cancer group. Am J Epidemiol 1992; 136: 1175–83.

  1. 193 CusidoM, FabregasR, PereBS, et al. Ovulation induction treatment and risk of borderline tumors. Gynecol endocrinol 2007; 23(7): 373–6.
  1. BrintonLA, moghissiKS, ScocciaB, et al. Ovulation induction and cancer risk. Fertil steril 2005; 83: 261–74.
  1. AyhanA, SalmanMC, CelikH, et al. Association between fertility drugs and gynaecologic cancers, breast cancer and childhood cancers. Acta Obstet Gynecol Scand 2004; 83: 1104–11.
  1. SacksPC, SimonJA. Infectious complications of intrauterine insemination, a case report and review of literature. Int J Fertil 1991; 36: 331–9.
  1. WongPC, BalmacedaJP, BlancoJD, et al. Sperm washing and swim up techniques using antibiotics remove microbes from human semen. Fertil Steril 1986; 45: 97–9.
  1. KerinJF, QuininP. Supercervical placement of spermatozoa: utility of intrauterine and tubal insemination. In SoulesMR (Eds): controversies in reproductive endocrinology and infertility. 1989, Elsevier.  New York,
  1. AllenNC, HerbertCM, MaxsonWS, et al. Intrauterine insemination a critical review. Fertil Steril 1985; 44: 569–80.
  1. LalichRA, MarutEL, PrusGS, et al. Life table analysis of intrauterine insemination pregnancy rates. Am J Obstet Gynecol 1988; 158: 980–4.
  1. DodsonWC, HaneyAF. Controlled ovarian hyperstimulation and intrauterine insemination for treatment of infertility. Fertil Steril 1991; 55: 457–67.

Determinants of Success and Optimizing Success of IUI13

196The efficacy of treatment for infertility disorders can be easily assessed by pregnancy rates per treatment cycle. The pregnancy rates per cycle with IUI have been reported between 8–22% in various studies1. However it is difficult to evaluate the success rates as there are different age groups, stimulation protocols, sperm preparation techniques, insemination techniques etc. and the successful outcome depends on a number of factors. Couples with cervical factor and unexplained infertility as well as those undergoing donor IUI demonstrate higher pregnancy rates. Most patients who get pregnant with IUI do so within the first 4 cycles. These many cycles can be completed within a year's time. Therefore, it is advisable to recommend IVF/ICSI to patients who fail to become pregnant after one year of IUI therapy.
 
FACTORS INFLUENCING PREGNANCY RATES WITH IUI
These factors are discussed and listed in Table 13.1 and elaborated briefly below:
 
Laboratory Factors
 
General factors
 
Female factors
Age: Age of the women is the single most important determinant of success. Success rates decrease after 35 years of age and fall, dramatically after 40.
Cause of infertility in female partner: Cause of infertility in the female partner affects the success rates with IUI. Unexplained infertility and anovulation have better results compared with other etiologies.2 Endometriosis and tubal factor infertility have a negative impact (Table 13.2).
198Controlled ovarian stimulation: Numerous studies have shown that better results are obtained with superovulation compared with natural cycle. Results also vary with the type of stimulation protocol used. Stimulation with gonadotropin gives better results compared with clomiphene citrate. In a study conducted by Afnan M, HMG stimulation gave better results (9%) compared with CC (4%) and natural ovulatory cycle (2%).4
Letrozole produced comparable pregnancy rates to CC.5 Addition of letrozole to FSH decreased the dose of FSH required for stimulation, however, the pregnancy rates remained the same, making it more cost-effective compared from FSH alone.6
 
Concomitant use of GnRH Agonists with Superovulation and IUI
The GnRH agonists act by down regulation to bring down raised levels of LH which interfere in folliculogenesis and also prevent premature LH surge. There is a definite advantage of combining GnRH agonist with HMG/FSH in stimulating patients undergoing IVF/ICSI, popularly known as the Long protocol. However, the advantage of using such a protocol for IUI is less clear as they prolong the treatment and increase the cost as higher doses of gonadotropins is required. Also, many studies have shown no difference in the outcome (Flow chart 13.1).7
 
Concomitant use of GnRH Antagonist with Superovulation and IUI
GnRH antagonists prevent premature LH surge and luteinization and thus improve pregnancy rates. The rate of premature LH surge is decreased from 35% to 7%.8
199
Flow chart 13.1: Protocol for optimizing the success rate
It has the advantage of having a convenient, small and acceptable dosage and also helps scheduling the IUI, avoiding the weekends. The GnRH antagonist is given in the dosage of 0.25 mg SC when leading follicle is > /= 14 mm. This is given daily till the hCG is given for ovulation.
200Number of preovulatory follicles: Multifollicular development results in an increased number of mature oocytes and a better quality endometrium and luteal phase thereby improving fertilization and implantation rates. Pregnancy rates increase with the number of mature preovulatory follicles. In a study conducted by Huttunen et al, the highest pregnancy rates were observed when there were three preovulatory follicles.9
Endometrial response: The endometrial thickness is an important factor for a successful outcome. Usually no pregnancy occurs when ET is less than 6 mm. Trilaminar appearance of the endometrium reflects good endometrial receptivity.
 
Male Factors
 
Age
In males the age does not seem to hamper the pregnancy rates as the aging process is slower in men compared to women.10 However, there are higher sperm abnormalities observed after the age of 50 years.
 
Semen parameters
Sperm count, motility and morphology are important factors for success. A positive correlation is seen between pregnancy rate and total motile sperm count. A significant rise in pregnancy rate is seen with post wash TMS (Total Motile Sperms) of more than 5 million per ml. Percentage motility correlates better than total motile sperm count.
IUI is reasonable first line therapy for patients with normal sperm morphology of more than 4%, provided other semen parameters are normal. In a study conducted 201by Shibahara et al, the pregnancy rate per IUI cycle were 3.8%, 18.5%, 29.9% in patients with sperm morphology with <4%, 4–9%, >9% normal forms.11 Couples with teratospermia, with <4% normal sperms or inseminate motile count of less than 1 million should be considered for ICSI.
 
Collection of semen
Sample should be processed and inseminated as soon as possible as longer intervals can lead to fluctuation of temperature, change in pH of medium or contamination which can affect quality of semen and thus the success of the procedure. Semen processed within 30 minutes after collection and IUI performed within 90 minutes of collection results in higher pregnancy rates.
 
Sperm processing methods
There is no consensus on sperm processing methodologies for IUI. A recent Cochrane analysis observed no difference between pregnancy rates or miscarriage rates for swim up vs. density gradient and centrifugation technique.12
 
Cryopreserved samples
Cryopreservation of sperms leads to 30–40% decrease in sperm motility and 10–15% decrease in pregnancy rates compared with fresh semen. This happens due to loss of membrane integrity and a concomitant decrease in metabolic rate due to freezing.13
 
Common Factors
 
Duration of Infertility
Pregnancy rates fall with increasing duration of infertility. Although the precise duration of infertility after which the 202success rates decrease cannot be defined, IUI should be recommended with caution to patients with long standing infertility.
 
Number of IUI Cycles
It has been seen that the pregnancy rates per cycle are nearly the same for the first 3–6 cycles, after which there is a fall. Most pregnancies occurred within the first four treatment cycles, favoring a maximum of four IUI cycles before switching to more advanced ART.9
 
Number of Inseminations Per Cycle
There is no consensus as to how many times insemination should be done per cycle. A recent Cochrane meta-analysis showed that performing double insemination does not increase the success rates compared to single insemination. It is usually performed 36 hours after hCG injection.14
 
Type of Catheter
No significant differences are noted for clinical pregnancy rates and live birth rate with different catheters. IUI with soft catheter was little difficult and with firm catheter little more patient discomfort is noted. Catheter choice doesn't seem to have a detrimental effect on success rates of IUI.15
 
Laboratory Factors
  1. Setting up proper laboratory with good quality control
  2. Standardized equipment: A good centrifuge and incubator, preferably CO2 incubator for processing the semen sample.
    203
  3. Maintenance of good air quality in the laboratory.
  4. Readymade standardized culture media.
  5. Utilizing disposable, sterile and toxicity tested plastic semen containers, tubes, pipettes and occasionally non-toxic condoms.
  6. Avoidance of major chemicals such as methyl alcohol, formalin, powdered latex gloves, etc in the lab and during the insemination procedure.
  7. Cleaning laboratory walls and floors with dilute chlorhexidine at the end of the day.
  8. Proper collection, liquefaction, processing of sample.
REFERENCES
  1. CohlenBJ, te VeldeER, van KoojiRJ, et al. Controlled ovarian stimulation and intrauterine insemination for treating male subfertility: a controlled study. Hum Reprod 1998; 13: 1553–8.
  1. KhalilMR, RasmussenPE, ErbK, et al. Homologous IUI. An evaluation of prognostic factors based on review of 2473 cycles. Hum Reprod 1999; 14: 698–703.
  1. MentanaroGM, KrugerTF, CoetzeK, et al. Stepwise regression analysis study to study the male and female factors impacting on pregnancy rates in an IUI programme. Andrologia 2001; 33: 135–41.
  1. AfnanM. The pregnancy rates with intrauterine insemination in superovulating cycles employing different protocols (clomiphene citrate (CC), human menopausal gonadotropin (hMG), CC + HMG) and in natural ovulatory cycle. J Pak Med Assoc 2004; 54: 503–5.
  1. KuSY, SuhCS, KimKC, et al. Use of letrozole vs. clomiphene citrate combined with gonadotropins in IUI cycles: a pilot study. Fertil Steril 2006; 85: 1774–7.
  1. BedaiwyMA, FormanR, MousaNA, et al. Cost effectiveness of aromatase inhibitor co-treatment for Controlled Ovarian Stimulation. Hum Reprod 2006; 21: 2838–44.

  1. 204 DodsonWC, HaneyF. Controlled ovarian stimulation with intrauterine insemination for treatment of infertility. Fertil Steril 1991; 55: 457–67.
  1. AllegraA, MarinoA, CoffaroF et al. GnRH antagonists induced inhibition of premature LH surge increases pregnancy rates in intrauterine insemination stimulated cycles. A prospective randomized trial. Hum Reprod 2007; 22: 101–8.
  1. HuttunenSN, TomasC, BloiguR et al. IUI treatment in subfertility. An analysis of factors affecting outcome. Hum Reprod 1999; 14: 698–703.
  1. MathieuC et al. Cumulative conception rates following intrauterine artificial insemination with husbands spermatozoa: influence of husband age. Hum Reprod 1995; 10: 1090–7.
  1. ShibaharaH, ObaraH, Ayustawati et al. Prediction of pregnancy by IUI using CASA estimates and strict criteria in patients with male factor infertility. Int J Androl 2004; 27: 63–8.
  1. BoomsmaMV, HeinemanMJ, CohlenBJ et al. Semen preparation techniques for IUI. Cochrane Database Syst Rev 2007; 17(4): CD004507.
  1. AlfredsonJH, GudmunnsonSP, SnaedelG. Artificial insemination with donor with frozen semen. Obstetr Gynecol Survey 1983; 38; 305–13.
  1. CautineauAEP, HeinemanMJ, CohlenBJ. Single vs. double IUI in stimulated cycles for subfertile couples. Cochrane Database Syst Rev 2003; issue1. art no: CD003854.
  1. AhmedMA, RaganaTM, HeshamG, et al. IUI catheters for assisted reproduction: a systematic review and meta-analysis. Hum Reprod 2006; 21: 1961–7.

Donor Insemination14

206Donor IUI or artificial insemination refers to the procedure of deposition of washed and concentrated donor sperms in the uterine cavity of the woman through artificial means.
 
INDICATIONS1
Indications for donor insemination are many. Male factor infertility is the commonest of them.
  • Azoospermia— Absence of sperms in the ejaculate
  • Severe oligoasthenospermia, severe teratozoospermia — Couple with severe male infertility, unable to opt for ICSI due to cost constraints.
  • Genetic abnormalities—Males with Y chromosome deletion, carrier of mutations incompatible with life.
  • Consanguinous marriages leading to early miscarriages due to autosomal recessive disorders.
  • Rh incompatibility.
  • Female without a male partner.
 
DONOR WORK-UP
The prospective donor should be a healthy young man, between 21–45 years of age with no personal and family history of illness. They should be willing to donate voluntarily. It is preferable to choose a donor with proven fertility. No owner, operator, laboratory director or employee of the facility providing therapeutic donor insemination (TDI) should serve as a donor in that practice.
Before acceptance, and every 6 months while remain­­ing as the active donor, donors should undergo a complete 207physical examination, and should be declined if there is any evidence of STD's, nonmedical percutaneous drug use, disseminated lymphadenopathy, oral thrush or unexplained jaundice.
Donors are screened for sexually transmitted infections like syphilis, HIV infection, and hepatitis B and C infection. Blood group and screening tests for potential genetically transmittable diseases, is also included in the initial work-up.
The semen is analyzed according to the WHO manual method for analysis and accepted only if it is normal (Table 14.1).
Table 14.1   Minimal semen parameters recommended for donors (ASRM guidelines)2
Volume
>2 ml
Sperm motility
>50% moving actively
Sperm concentration
>20 × 106 motile sperms / ml
Sperm morphology
Normal range
Cryo survival
>50% of initial motility
Donor sample can be fresh from an investigated donor, or, a sample can be acquired from the semen bank.
The advantages of using frozen semen sample are:
  • Quarantine of donor semen till appropriate testing can be completed.
  • Ease of scheduling insemination when ovulation is optimal.
  • Availability of consistent quality of samples.
  • Sequential insemination with the same donor can be achieved.
    208
The disadvantage of frozen sample is that the timing of the IUI has to be very precise as the life span of the thawed specimen is only about twelve hours.3
According to ASRM, after donation all sperm samples should be quarantined for 180 days and specimens should be released only if the repeat testing for infectious diseases are negative. The use of fresh semen can only be justified in case of sexually intimate couples.
 
Psychological Issues and Counseling1
The decision to proceed for donor insemination is complex and many psychological issues are related to it. Both the partners may feel the stress and emotional turmoil. Psychological counseling should be offered to them prior to the procedure and if needed, even after the successful donor insemination. The couple should be counseled regarding:
  • The use of donor semen—The anonymity of the donor is maintained. The physical attributes and blood group of the donor can be matched with the husband. The couple needs to sign an informed consent regarding the use of donor sperms.
  • The risk of hyperstimulation—The patient is counseled about the risk of hyperstimulation and eventual cancellation of the IUI cycle and its conversion to IVF/ICSI.
  • The chances of multiple pregnancy—The chances of multiple pregnancy are higher than in normal conception due to the stimulation of the ovaries, to release more than one mature oocyte.
    209
  • The risk of miscarriage—The risk of miscarriage is higher in older women, PCOS cases and those with multiple pregnancies.
 
Consent
It is mandatory for the couples to sign the consent prior to the procedure. The consent of the husband is mandatory.
 
Legal Aspects and ICMR Guidelines for Donor Insemination
The ICMR has formulated guidelines for donor insemination and ART clinics in 2005.1
 
Responsibilities of the Clinic
 
Confidentiality
Any information regarding the donors or clients must be kept confidential.
 
Avoiding a Mixing of Semen Samples
Under any circumstances, semen from two individuals must never be mixed.
 
Limitations to Donor Use
According to ASRM, it is difficult to provide the precise number of times that a given donor can be used. It has been suggested that limiting the single donor to no more than 25 births would avoid any significant increased risk of inadvertent consanguinous conception.
According to ICMR, sample from one donor should not be used for more than 75 times.
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Record Keeping
A detailed record of all donor sperm and the couple using them should be maintained for at least 10 years. The records must be transferred to a central repository maintained by ICMR.
 
Known Donor
Use of any donor sperm known either to the wife or husband shall not be permitted under any conditions.
 
Legal Issues in Marriage
Without consent of husband, donor insemination can form ground for divorce.
REFERENCES
  1. The assisted reproductive technologies (regulation) bill, 2008 ICMR,  New Delhi.
  1. ASRM practice guideline: 2008 guidelines for gamete and embryo donation. Fertil Steril 2008; 90:S30–44.
  1. Martinez,O. Lazcano, M.Chazaro, et al. Intrauterine insemination using donor sperm: a report of 2 years of experience. Fertil Steril 2008; 90: S463.

Sperm Cryopreservation15

212Cryopreservation of human spermatozoa is widely used in many assisted reproduction centers and andrology laboratories. It was first reported in 1954.1 It involves initial contact of the spermatozoa to cryoprotectant, and then cooling them to subzero temperature. These are then stored in containers of liquid nitrogen at −196°C till required.
 
INDICATIONS OF CRYOPRESERVATION
Semen can be preserved for the use of individual himself, known as ‘autologous sperm banking or client depositor sperm banking’. It is routinely used in variety of circumstances as:
  • In patients of malignancy prior to surgery or chemo-radiotherapy
  • For persons who work in places with risk of radiation hazard
  • As fertility insurance in men undergoing vasectomy
  • For storage of sperms retrieved by electroejaculation or surgically retrieved sperms via PESA or TESA
  • For individuals who are frequent travelers or due to anticipated absence on the day of insemination.
  • In anticipated performance failure on the day of insemination.
When the semen is stored from fertile donors to be used by a third party, it is known as donor semen banking. Donor is screened both genetically and medically for infections such as HIV and hepatitis B and C. Semen is stored only after confirming seronegativity.213
 
PRINCIPLES OF CRYOPRESERVATION
Cryopreservation is a process, which maintains cell life for an extended period at remarkably low temperatures.
Cryopreservation aims to minimize cellular injury and cell membrane damage associated with intracellular ice formation, water removal and exposure to low temperatures. The principle of cryopreservation is to remove intracellular water before freezing to the extent that it does not remain a threat to the viability of cell, control cell volume and cooling rate.
The intracellular and extracellular movement of molecules depends on:
  • The cryoprotectant solution used
  • The rate at which the cells are cooled
  • The temperature at which the sample is plunged into liquid nitrogen
  • The warming rate
  • Cryoprotectant removal after thawing.
Cells undergo several volume changes as they undergo cryopreservation and thawing. At least half of the motile sperms will sustain cryo-injury when subjected to cryopreservation and thawing.2 At first when the cryoprotectant is added a rapid shrinkage in the cell size occurs as the water leaves out of the cell because of hyperosmotic extracellular solution. The cell gradually regains its original volume as the cryoprotectant agent permeates the cell. Presence of cryoprotectant results in continuous drop in freezing point of the remaining liquid and an increase in extracellular osmotic pressure. Additional intracellular water is drawn out of the cell in response to increasing 214extracellular osmotic pressure, leaving the cell dehydrated and reducing the risk of lethal intracellular ice formation.3
 
Cryoprotectants
These compounds are used to achieve the required intracellular dehydration. These are of two types:
  • Permeating: They enter inside the cell and displace the water molecule out of the cell, for example, dimethyl sulphoxide (DMSO), propylene glycol, glycerol.
  • Nonpermeating: That remain out of the cell and draw out the intracellular water by osmosis, e.g. sucrose, raffinose and glycine.
Glycerol is a important compound used to prepare the cryoprotectant, which is necessary for sperm freezing. It is superior to DMSO or ethylene glycol as a cryoprotectant. Various studies have shown that glycerol increases the motile sperm cryosurvival to an average of 50%, well above the < 20 percent reported without addition of glycerol.4,5
To store the gametes a combination of compounds is used, e.g. sucrose and one of the permeating cryoprotectant.
Cryoprotectant solutions have other compounds also known as extenders, which have protective effects on the cells during freezing and thawing, for example, citrate, egg yolk and zwitter ionic buffers. Egg yolk is used as an extender in sperm cryopreservation media only. The various lipid constituents of egg yolk bind to spermatozoa membrane and affect its fluidity by altering the cholesterol/phospholipids ratio. These changes may cause 215sperm to undergo changes associated with capacitation, stabilize the acrosomal membrane, and reduce the incidence of spontaneous acrosome reaction.
The components of cryoprotectant media are (Fig. 15.1):
  • Nutrients
  • A buffer
  • A cryoprotective agent
  • An antibiotic.
 
Cooling and Warming Rates
Both cooling and warming rate influences cell survival. The optimum cooling rate should be one that allows enough water to leave the cell before freezing occurs. If freezing occurs before that the excess intracellular ice crystal formation may become lethal to cell. A relatively slow cooling rate allows excess water to leave the cell, resulting in extreme cell shrinkage and cell dehydration.6
During thawing, there should be controlled diffusion of cryoprotectant out of the cell and for the re-entry of the water molecules.
Fig. 15.1: Sperm freezing medium
216Rapid thawing may result in swelling and lysis of cell due to rapid influx of the water. The optimum warming rate depends on cooling rate. Sperms cooled at slow rate should be warmed slowly.
 
STEPS OF CRYOPRESERVATION
Cryofreezing of the sample can be done using controlled rate freezer or liquid nitrogen vapor (non-controlled rate freezing). Literature has revealed no differences in sperm cryosurvival or post-thaw motility. However, non-controlled rate freezing is simpler and less expensive. The choice may be opted by individual laboratory. At our center, we prefer to use liquid nitrogen vapor technique.
 
Noncontrolled Rate Cryopreservation (Liquid Nitrogen Vapor Cooling)
  • The sample is collected in a sample collection room within the premises to avoid contamination and temperature related changes.
  • Confirm the particulars of the patient.
  • After liquefaction, pipette out the sperm in a test tube and measure the total amount of the ejaculate. Do a semen analysis as per WHO standards.
  • Take an equal amount of sperm freezing media (SFM) in another test tube using a clean pipette. Ensure that both sample and freezing media are at 37°C.
  • Add the medium to the semen sample. It should be added drop by drop over a period of ten minutes and gently mixing after adding each drop. This step is essential to reduce the toxicity of cryoprotectant glycerol.
    217
  • Load the diluted semen into straws or cryovials labeled with patient's particulars and date and seal them. It is important to leave some space in the lower part of the straw to allow expansion of the solution during freezing.
  • Keep the filled straws or vials in a labeled aluminum cane.
  • Refrigerate the sample at 4°C for 15 minutes.
  • Pour liquid nitrogen in a thermocol box and allow equilibrating for 20 minutes.
  • Suspend the straws horizontally 4 cm above the nitrogen vapors for 30 minutes.
  • Finally, plunge it into liquid nitrogen cylinders (Fig. 15.2) and store at −196°C.
  • Record the position of the cane.
All storage containers should be stored in a secure room. Liquid nitrogen levels in the storage units should be checked regularly. At −196°C storage of sperms, even for a long period does not affect the survival rates, as there is virtually no movement of atoms or molecules.
 
Controlled Rate Freezing
It is recommended for freezing washed semen samples.
  • The vials are placed in a controlled rate freezer and preprogrammed freezing cycle is initiated.
The start temperature is 20°C At first it cools at 5ºC/min to 4°C
Fig. 15.2: Liquid nitrogen containers
218Subsequently at 10°C until −70° C
  • The samples are held for 10 minutes.
  • The vials are put into aluminum canes.
  • They are plunged into liquid nitrogen cylinders.
 
Thawing
  • Remove the required vial and allow it to thaw at room temperature for 20 minutes in the laminar flow hood. Alternatively, they can be thawed at 37ºC in a water bath for 5 to 7 minutes.
  • Clean the external surface with absolute alcohol and allow the alcohol to vaporize.
  • Transfer the contents of the vial in a clean test tube.
  • Add 2 ml of flushing media to reduce the toxic effects of glycerol.
  • Do a post thaw analysis?
  • Appropriate method of sperm preparation is applied immediately and final sperm count as well as motility is assessed before using for ART procedures.
219
REFERENCES
  1. ShermanJK. Freezing and free drying of human spermatozoa. Fertil Steril 1954; 5: 357–74.
  1. QuinnPH. Principles of membrane stability and phase behavior under extreme conditions. J Bioenerg Biomemb 1989; 21: 3–19.
  1. FarrantJ. Mechanism of cell damage during freezing and thawing and its prevention. Nature 1965; 205: 1284–7.
  1. HammittDG, WalkerDL, WilliamsonRA. Concentration of glycerol required for optimum survival and In vitro fertilization capacity of frozen sperms is dependent on cryopreservation medium. Fertil Steril 1988; 49: 680–7.
  1. CritserJK, Huse-BendaAR, et al. Cryopreservation of human spermatozoa. The effect of cryoprotectants on motility. Fertil Steril 1988; 50: 314–20.
  1. TylerJPP, KimeL. Temperature change in cryo-containers during short exposure to ambient temperatures. Hum Reprod 1996; 11: 1510–2.
IndexAAbdominaldistension girth pain Abnormalcervical canal , postcoital test sperm morphology Abortion Absence of sperms in ejaculate Accidental insemination with wrong sample Acid phosphatase level Acutelyanteverted uterus retroverted uterus Adnexal torsion Advantages ofantagonist protocol semen preparation using Makler chamber Agglutination Aggregation Albumin/hydroxyethyl starch Allergic reaction , Allis forceps Anovulation Anterior wall retractor Antimullerian hormone Anti-sperm antibodies , Antral follicle count ARDS Ascites Aspermia Assessing follicular maturity Asthenospermia Asthenozoospermia Atherosclerosis Automatic sperm analyzer systems Avoiding mixing of semen samples Azoospermia , , BBaseline scan of ovaries Basic steps of insemination BD syringe Bench space Bilateral tubal block Body mass index CCabergoline CASA systems Cause of infertility Centrifuge , with settings , Chamber description Chances of multiple pregnancy Checking for liquifaction Choice ofgonadotropins insemination cannula Classification of OHSS Clomiphenecitrate , , challenge test with gonadotropins with gonadotropins CO incubators Coagulation profile Collection ofsemen , , specimen Complications ofIUI OHSS Compound microscope Concomitant use of GnRHagonists with superovulation and IUI antagonist with superovulation and IUI Confidentiality Congenital absence of vas Conicalbottom tubes tube Consanguinous marriages Consent Control of sperm entry into upper genital tract Controlledovarian stimulation rate freezing Cooling and warming rates Correction ofcirculatory volume electrolyte imbalance Counting method Cryocans Cryopreservation media tCryopreservedsamples semen Cryoprotectants Culture media Cumulus oophorus in follicle Cusco's speculum DDebris Decreased renal function Decreasing dose of hCG Delaying hCG Density gradientcentrifugation method , , Detecting LH surge Determinants of success and optimizing success of IUI Diabetes mellitus Difficult IUI Digitalheater heating block Directintraperitoneal insemination overlay method Disposables required in IUI laboratory Diuretics Donor insemination Drug therapy Duration ofinfertility , support Dydrogesterone EEctopic pregnancy , Ejaculatoryducts or seminal vesicles dysfunction , fail Electroejaculation Endometrialbiopsy grading response , thickness Endometriosis , , , Endometrium , Epididymal sperm aspiration Epithelial cells Equipments , Evaluation offemale partner male partner Excessive pus cells Exposing cervix Extended letrozole therapy Factors influencingdose of gonadotropins pregnancy rate with IUI FFailed emission Fallopian tube sperm perfusion Faulty collection Features of mature endometrium Female without male partner FlushingIUI cannula media for IUI Follicle aspiration French protocol Frequent ejaculation Fructose level GGenetic abnormalities Genital tract infection Glass wool filtration Global vascular indices GnRHagonist , analogue in combination with gonadotropins Golan's classification of OHSS Gonadotropins , , with GnRH analogues with GnRH antagonists Gynecomastia HHair distribution scoring Heating block Hemoconcentration Hemocytometer HEPES buffered culture media Hereditary disease in father High resistance uterine artery flow Highly viscous semen HIV infection , Hormonal investigations Human chorionic gonadotropin , , Hydrothorax Hyperechogenic endometrium Hypoechogenic endometrium Hypogonadotropic hypogonadism Hypospadias Hypospermia , Hypothalamic pituitarydysfunction failure Hysterosalpingogram IIncubator , Indications ofcryopreservation hospitalization IUI Infection causing abnormal prostatic function Initiation of capacitation Insemination procedure technique with donor sperms with husband's frozen semen Insertion of IUI cannula through cervix Instrument trolley for IUI Instruments for IUI Intercavernosal injection Intracytoplasmic sperm injection Intraovarian resistance index Intrauterine insemination , , , Inverted microscope ISAS IUIcannula with syringe catheter lab room Ivos sperm analyzer LLabelling ofconical tube semen sample tubes Laboratory design Laminar flow cabinet hood Layering of density gradient with semen Legal issues in marriage Letrozole , , and ovulation induction step up protocol therapy with gonadotropins , Liquefaction Liquid nitrogencontainers t, vapor cooling Liverdysfunction enzymes function tests LoadingIUI catheter Makler to check sperm count , of semen sample in IUI catheter Lowresistance ovarian stromal flow sperm counts volume Lubeck protocol Luteal phasedefect support in IUI Luteinized unruptured follicle Luteinizing hormone MMaintainingintravascular volume renal function Maklercatheter chamber , , , insemination device instrument Male subfertility Mantoux test Manual sperm analysis systems Mature follicle Measuring functional uterocervical length Media , , Meiosis Methods of assessment of sperm concentration Micronized progesterone Microscope , Mild OHSS Minimal semen parameters recommended for donors Monitoringendometrium follicular growth in intrauterine insemination of follicular growth and endometrial development Morphology index Motile sperm count Multipleinfertility etiologies pregnancies , , sclerosis NNatural progesterone Necrozoospermia Neubaur chamber No pregnancy despite ovulation Noncontrolled rate cryopreservation Non-infective salpingitis , Normaloocyte maturation semenanalysis and CASA parameters as suggested by WHO morphology Number ofantral follicles inseminations per cycle , IUI cycle , preovulatory follicles , Nutrition of sperms OObstruction of ejaculatory tract Odor Oligospermia Oligozoospermia Oocyte maturation Open centrifuge fOvariancysts enlargement tfailure hyperstimulation syndrome , , , , induction , reserve stimulation , , , , protocols for ovulation induction Overlay method , Overlaying of pellet with media Ovulationafter hCG injection in controlled ovarian hyperstimulation induction , in IUI trigger and timing of insemination PPain Papaverine , Paracentesis of hydrothorax PCOS Pelletafter second centrifugation drop Pelvicmass sonography surgery Penile vibrator Percutaneousepidydimal sperm aspiration , vasal sperm aspiration Phenothiazines Phenoxybenzamine Phentolamine Pipettes Placing tubes in centrifuge Poor semen parameters Post wash sperm count Post-insemination instructions Post-IUI instructions Powder free gloves Pregnancy contraindicated in female partners rate , Preovulatory scan Prerequisites for IUI Prevention of thrombosis Principles of cryopreservation Procedurefor IUI room Progestogens Prostatic dysfunction Protection of sperms from vaginal acidity Protocol for optimizing success rate Psychological issues and counseling RBCs Recent advances in semen analysis RecombinanthCG , LH , Reconstitution of pellet Refrigerator , Removal of supernatant Renaldysfunction failure function tests Retrograde ejaculation , Rh incompatibility Rhesus isoimmunization Risk factors for OHSS Risk of hyperstimulation Risk ofmiscarriage ovarian cancer , Role ofantibiotics Doppler in intrauterine insemination sonography in difficult situations Round bottom tubes SSamplecollection room overlaying Selection of sperms based on motility Semenanalysis and culture collection and sample assessment containers parameters , preparation techniques for IUI sample Seminal plasma Serial serum estradiol levels SerumAMH estradiol levels Setting up of IUI laboratory Severeoligoasthenospermia teratozoospermia Severely subnormal semen parameters Sexually transmitted disease Short abstinence interval Simple wash Sims speculum Soft artificial insemination catheters Spermconcentration count counting chamber , cryopreservation freezing medium morphology , motility , preparation , media processing methods , reservoir function select method viability Spermatids Spermatozoa , Spinal cord injury Sponge holder Stenosed cervix Steps of cryopreservation Steroids Stimulated cycle Streptococcus Subnormal semen parameters Swim uptechnique , without centrifugation TTechniquesfor evaluation of sperm motility of intrauterine insemination Tenaculum Teratozoospermia , , Termination of pregnancy Test tube stand Testicular sperm extraction Testsbefore ovarian stimulation for antisperm antibodies for ovulation to rule out tuberculosis Thawing Therapeutic donor insemination Timing of insemination Tomcat IUI catheter Totalovarian stromal area ovarian volume Transurethral resection of prostate Transuterotubal insemination Trauma and bleeding Trichomonas vaginalis Triple line endometrium , Tubalfactor infertility pathology Tuberculin syringe Tygerberg-Kruger strict criteria Types ofartificial insemination catheter , progesterone techniques UUltrasoundfollicular monitoring image of hyperstimulated ovary Unexplainedgenital tract bleeding infertility Upper genital tract Ureaplasma Urinary hCG Use of donor semen Uterocervical canal Uterus VVascularcomplications endothelial growth factor Vascularity index Vasectomy Vasomotor symptoms , Venous compression Very severe oligoasthenospermia Viscosity Volume of insemination WWallace catheter WBC count Withholding hCG ZZinc