Follicular Response by Using Letrozole Alone or Combination of Letrozole and Gonadotrophins in Patients Stimulated For Intrauterine Insemination

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Follicular Response by Using Letrozole Alone or Combination of Letrozole and Gonadotrophins in Patients Stimulated For Intrauterine Insemination

Introduction:

Throughout history, human societies were preoccupied by the process of procreation which they attempted to describe through art and religion. It is one of the major aspects of maintaining survival and perpetuation of the human race. In view of the entwined nature between sexuality, morality, and reproduction, fertility played a pivotal role in almost all religions on this Earth.

As described in the Bible, and in numerous historical documents, infertility problems have played a central role in the relationship between man and woman, since time immemorial, and both laws and religions have been used to regulate right and wrong in relations between married couples who face infertility (divorce, multiple marriages among Muslims, adoption, suicide, etc).

The number of subfertile couples is increasing numerically in an expanding global population, even if infertility per se does not increase. Unfortunately, factors such as environmental pollution and sexually transmittable diseases tend to further exacerbate the problems in certain parts of the world, especially in developing countries, where they are predominantly associated with secondary infertility.¹

Bangladesh is a developing country with an exploding population of 158 million, a total fertility rate of 2.74 per thousand live births and a growth rate of 1.292 %.² In spite of that, there are a large number of couples who remain childless for years together. Sub fertility in a woman does not claim her life or maim her physically, but she suffers from psychological trauma of being unable to fulfil her biological role of being a mother, which is more pronounced in Asian and African countries. It becomes worse if she has to bear the humiliation on behalf of her husband, who could be the cause of infertility.

Prevalence of infertility in developing countries is difficult to assess due to inconsistencies in defining infertility. Between 8% – 12% of couples around the world have difficulty in conceiving a child at some points in their life.³

Data from U.S. National survey of family growth indicate that the prevalence of infertility was 13.35% in 1965, and 13.9 % in 1988.⁴ Although the overall prevalence of infertility in USA has not been changed since 1965, but the demand for infertility care has increased significantly in the last few decades. Data from U.S. National survey of Family Growth-1995- showed that 13% had sought medical attention for infertility in their lifetime.⁵

The Indian reports, both from the institute for Research in reproduction, Mumbai and from Mehta et al, Bangalore ⁶ seem to agree that the declining trend of semen quality over the years is responsible for increased male factor infertility.⁶

In a study of rural population of Bangladesh 3.2% of married women of reproductive age have primary infertility and 3% have secondary infertility.⁷ Majority of infertile women (40%) believed this to be their fate and 33% accused themselves for infertility.⁷

Prior to 1960s, infertility was almost exclusively a diagnostic statement with no treatment possibilities. It is well known that mechanical, hormonal, and psychological factors, in either the female or male partner could be responsible for infertility. Sometimes, more than one of these factors is involved, which thus become obvious during the workup of the couple, but reasonably often, no explanation for the infertility could be found, a condition referred to as ‘unexplained infertility. It was believed that perhaps, more diffuse psychological factors played a predominant role in these cases.

Technical innovations in the laboratory and by the pharmaceutical industry have helped to promote the success rate of infertility treatment. The introduction of invitro fertilization & embryo transfer (IVF-ET) in 1980s has made it possible for the couple to take advantage of this technology. Winning of Nobel Prize of the year 2010 for the 1^st successful IVF was the golden recognition of this method by the world. Though Vatican City & activist Muslims are yet against IVF-ET, but for some subfertile couples, it is their only hope.

The true clinical meanings of terms such as ‘Pregnancy rates’ or ‘the chances of success’ must be explained thoroughly while counselling a couple – which can be explained in medical science by words – fecundability and fecundity. Fecundability refers to the probability that actions taken during one menstrual cycle will result in a pregnancy. Fecundity is the possibility that actions taken during a single menstrual cycle will result in a live birth. Fecundity in women at the age of 20 is approximately 15% to 20% per cycle – whereas the fecundity in infertile couples can be as low as 1% to 3%. A marked decrease in fecundity with advancing age has been documented. Data from studies have suggested that fertility of women generally peaks between ages 20 and 24.⁸

WHO task force on the diagnosis and treatment of infertility in 1992 divided the infertility issue into male factors and female factors. The actual percentages of the individual factors that are found to be the primary cause of infertility vary widely between studies.⁹However in a broad Meta analysis of more than 20 trials studying infertile couples, the following primary diagnosis was found: Disorders of ovulation (27%), abnormal semen parametres (25%), abnormalities of the fallopian tube (22%), unexplained infertility (17%), endometriosis (5%) and others (4%).¹⁰

In couples of idiopathic or unexplained infertility, Assisted Reproductive Technologies may offer a non specific enhancement of cycle fecundity. The number of assisted reproductive technologies available as well as their use has dramatically increased since the birth of Louis Brown in 1978 from IVF-ET.

Controlled ovarian hyper stimulation combined with IUI of capacitated sperm (COH-IUI) has recently been used to treat a subset of couples infertile in the absence of mechanical compromise of the pelvic viscera, in whom no other efficacious treatment options exist .^{11, 12, 13} This approach has four potential advantages over simply awaiting the infrequent spontaneous conception, that is expected management. These include (1) increasing the number of oocytes available for fertilization; (2) increasing the levels of follicular and lutial phase gonadal steroids; (3) optimising the likelihood of gamete interaction with viable oocytes and sperm virtually guaranteed to be simultaneously present in the female genital tract; and (4) providing large numbers of capacitated sperm at the site of fertilization in the distal fallopian tubes. Many of these features are common to the more invasive oocyte retrieval techniques such as IVF-ET, gamete intra fallopian transfer (GIFT), zygote intra fallopian transfer (ZIFT).¹¹ The major difference with COH-IUI is that the normal fallopian tubes can be anticipated to function appropriately and no surgical procedure is required to collect oocytes. The initial suggestion that COH-IUI might be efficacious was based on the occasional pregnancies observed in women in IVF-ET programs when the oocyte retrieval was cancelled because of poor follicular recruitment and IUI substituted in an effort to salvage some benefit from the cycle. This therapy quickly became a popular therapeutic option because of the relatively lower cost, the avoidance of oocyte retrieval and laparoscopy and as cycle fecundity comparable with the other assisted reproductive technologies.

IUI is the best-studied and most widely practiced technique for mild to moderate male factor infertility. In a review, Hamburg and Insler¹⁴ concluded that, taking into account efficacy, complication rates and cost of infertility treatment women with hypogonadotrophic hypogonadism or polycystic ovary syndrome (PCOS) should be offered acceptable methods of ovulation induction and that couples with ‘unexplained’ or ’multifactorial subfertility’ should be exposed to controlled ovarian hyperstimulation with IUI (COH IUI) and only after the failure of these therapies should be offered invitro fertilization (IVF).¹⁴ To perform COH with IUI, at least one patent tube and some ovarian function in female and motile sperm in maleshould present.¹⁵ IUI is a fertility enhancing procedure in which sperms are prepared by washing, swim up and swim down techniques and by use of density gradients.¹⁶ The washing procedures are necessary to remove prostaglandin, infectious agents, antigenic proteins, nonmotile spermatozoa, leukocytes, and immature germ cells. Concentrated germ cells are inseminated directly into a woman’s uterus through the cervix. Intrauterine insemination increases the number of potent sperm by decreasing the formation of free radicals after sperm preparation, in the uterus and fallopian tubes- where fertilization takes place. The final result is an improved fertilizing capacity of the sperm in vitro and in vivo fertilization. ¹⁷ The use of washed sperm reduces painful uterine cramps, collapse and infection. ¹⁶

Controlled ovarian hyperstimulation (COH) has got the primary cycle fecundity enhancing effect due to the increase in the number of oocytes released in a given cycle for ovulation induction. For controlled ovarian stimulation in IUI cycles many ovulation induction protocol have been devised. Such as Clomiphene citrate, Aromatase inhibitor (Letrozole) alone or in combination with Gonadotrophins (hMG or rFSH), human chorionic Gonadotrophins (hCG); and the use of Gonadotrophin releasing agonist or antagonist combined with Gonadotrophins. hCG is usually used at the end of the stimulation phase to achieve final maturation of the oocytes.¹⁶ No well designed comparative trials have been performed to determine if one regimen is superior to another. By Clomiphene citrate (Cc), though follicular development is higher, but due to the potentially adverse effect on the oestrogen-responsive tissue in the genital tract, such as the cervical mucus, endometrium, and fallopian tubes, it yields a somewhat lower cycle fecundity than when the COH is induced with hMG., Letrozole hMG may offer some advantages in selected couples, reducing the dose of hMG required to achieve optimal stimulation. Letrozole stimulates an early follicular rise in the endogenous gonadotrophins, whereas hMG provides an increase in exogenous gonadotrophins later in the stimulation course. Injection hCG is administered at the end of induction for final maturation and luteal support. The addition of IUI to Letrozole increase fecundity in couples with unexplained infertility or surgically treated endometriosis.^{18, 19} As with almost all infertility, success is age related with pregnancy rates 3-4 fold greater in women under age 20. Ovarian stimulation with letrozole is associated with acceptable pregnancy rates compared with gonadotrophin (cumulative pregnancy rate per couple: 24% vs. 36%), ²⁰ with significant less cost, risk and patient inconvenience.

Rationale of the study

Bangladesh is a country of 158 million people. The number of subfertile couples seeking treatment is increasing day by day. Different infertility clinics are providing various modalities of treatment. IUI is one of the most popular treatments in our country. Mild to moderate male factor subfertility can be overcome by COH and IUI. Although there are a large number of studies on COH and IUI internationally, there are no studies in our country. The present study was conducted to determine the follicular response by different types of ovulation inducing drugs, the effectiveness of IUI and COH and its outcome and also to assess the cost effectiveness in a tertiary level infertility management centre in our country.

Hypothesis and objective Of the Study

Hypothesis:

Induction of ovulation by gonadotrophins along with letrozole gives better follicular development than letrozole alone in stimulated IUI cycles.

Objective of the study:

General objective:

To assess the follicular response by Letrozole alone or by combination of Letrozole and Gonadotrophins in patients stimulated for IUI.

Specific objective:

· To compare the follicular response by Letrozole alone or by combination of Letrozole and Gonadotrophins.

· To compare the follicular response by giving Letrozole alone in <30 years and >30 years patients.

· To compare the follicular response by giving combination of Letrozole and Gonadotrophins in IUI cycle in these two groups of patients.

Literature review

Causes of infertility and subfertility:

One of the broadest investigations conducted by WHO task force on the diagnosis and treatment of infertility in 1992²¹ has shown several significant differences in their findings depending on the economic environment of the populations studied. But the data was remarkably uniform.

1. Abnormalities of sperm production (Male Factor)

i) Abnormalities in hormonal support of spermatogenesis

ii) Abnormalities in spermatogenesis

iii) Obstruction of outflow tract

iv) Inability to perform successful coitus

2. Abnormalities in the ability to produce a fertilizable oocyte (Ovulatory Factor)

i) Gonadal disgenesis(Turner’s Syndrome)

ii) Premature ovarian failure

iii) Lutinized unruptured follicle syndrome

iv) Depletion of oocyte pool by:

a. Pelvic Surgery

b. Endometriosis

c. Infection, TB

d. Chemotherapy

3. Abnormalities of the female reproductive tract

i) Tubal Factor

ii) Peritoneal Factor

iii) Uterine Factor

iv) Cervical Factor

4. Abnormalities of implantation

i) Luteal phase defect

ii) Hyperprolactinaemia

iii) Insuline Resistance

iv) Embryo-endometrial factor

v) Abnormalties of early embryogenesis

5. Immunologic Factors

i) Autoimmune diease

ii) Anti-phospholipid syndrome

iii) Thrombophilias

iv) Recurrent pregnancy loss

Intrauterine insemination

An overview:

Artificial insemination is an assisted reproduction method that can be used to alleviate infertility in selected couples. The rationale behind the use of IUI is to increase the sperm density near the site of fertilization.²² It is generally accepted that IUI should be preferred to more invasive and expensive techniques of assisted reproduction (ART) and be offered as a first choice treatment in cases of unexplained and moderate male factor infertility and cervical factor infertility.²³ The benefit of IUI in male factor infertility has been accepted because IUI appears to result in higher pregnancy rates than natural intercourse or intracervical insemination (OR 2.20, 95% CI 1.43-3.39).Hamburg and Insler concluded that taking into account- efficacy, complication rates and cost effectiveness of treatment, women with hypogonadotrophic hypogonadism or PCOS should be offered acceptable methods of ovulation induction and that couples with unexplained and multifactorial infertility should be exposed to controlled ovarian stimulation with IUI and only after the failure of these therapies IVF should be offered.¹⁴

The use of IUI reduces the normal attrition of sperm as they ascend through the female genital tract and thus guarantees a large numbers of capacitated sperm at the site of fertilization in the distal ampulla of the fallopian tube.¹⁶ This is similar to what is accomplished in IVF-ET and GIFT, when higher sperm densities are used during the oocyte incubation (IVF), or placed retrograde in the fallopian tube (GIFT).²⁴ IUI is a distinctly non-physiological process. However, after the IUI, the sperm are physiologically capacitated by their passage through the genital tract.

IUI is a relatively simple procedure compared with IVF, and its popularity as a treatment option for certain diagnostic groups of infertile couples is increasing, since it is the intermediate between simple ovulation induction and the more “high tech” IVF. This is particularly so in developing countries, where the facilities for IVF may be limited, and the cost of treatment is a major issue. There are continuing discussions in the literature about whether or not IUI should be complimented by ovulation induction. Most practitioners are of the opinion that IUI with ovulation induction does increase the success rate, and many will initially try with Clomiphene Citrate or Letrozole, and move on to Gonadotrophins, if there is no success within a few cycles. The most appropriate time to move on from IUI to IVF is a matter of debate, but most agree that change should be made from no more than 4-6 cycles of IUI.

Before proceeding to IUI couples should undergo a complete assessment, which should include thorough medical history, physical examination and investigations. Tubal patency must be tested by laparoscopy or hysterosalpingography. It is essential that couples should receive adequate counselling prior to starting treatment, and also be assured of complete confidentiality, and informed consent should be taken. All the pros and cons of IUI should be disclosed to the couple.

Indication of IUI:

There are a number of indications for IUI using the husband’s or partner/donor’s (not accepted in our country) semen. The most common indications are listed below:

Effective:

·Male subfertility (oligozoospermia/asthenozoospermia/ terratozoospermia)

· Cervical Factor subfertility

· Immunology subfertility

· Ejaculatory failure (hypospadius, retrograde ejaculation)

· Unexplained infertility

· Mild Endometriosis

· Polycystic Ovary Syndrome (PCOS)

Timing of Insemination:

The ideal timing for IUI should be 36 to 40 hours after the ovulating dose of hCG, ensuring that sperm and oocytes are present simultaneously in the distal fallopian tubes.²⁵ The IUI can be performed 24 hours after the hCG trigger if there is any suggestions that a premature LH surge has occurred based on a slowing of the rate of rise of estradiol levels in serum.

A single randomized study using two inseminations at 24 and 48 hours after the ovulating dose of hCG compared with a single insemination 36 hours after the hCG found a higher cycle fecundity with multiple inseminations per cycle.²⁶

Risk of IUI ²⁷

Uterine cramp, lower abdominal pain, vesovagal attack as the seminal plasma contains prostaglandins.

Pelvic infection by E. Coli due to use of fresh semen

Veneral disease, HIV or hepatitis B or C.

Failure of the treatment, since pregnancy rates per cycleare reported at anywhere between 5% and 25%.²⁸

Induction of ovulation and controlled ovarian hyperstimulation:

Ovulation induction (OI) and controlled ovarian hyper stimulation (COH) are the corner stone of infertility management. The goal of fertility treatment is to achieve a high pregnancy rate using safe, simple and inexpensive treatment protocols. Clomiphene citrate (CC), offers a simple, inexpensive and relatively safe choice of drug for treatment, but unfortunately, the pregnancy rates are relatively low. Other drugs which are commonly used are Aromatase inhibitor (Letrozole), Gonadotrophins (rFSH and HMG), HCG. CC and hMG-hCG, Letrozole-hMG-hCG, Letrozole-rFSH-hCG, Gonadotrophins –hCG after pre-treatment with with a GnRHagonist. ¹²

The role of aromatase inhibitors in ovulation induction:

Pharmacology and Mode of action:

Letrozole are non steroidal selective aromatase inhibitors that have been approved for treatment for postmenopausal breast cancer since 1997.¹⁸ Aromatization of testosterone to estradiol and androstenedione to estrone is the last step in the biosynthesis of oestrogens. Letrozole inhibits the conversion of adrenally generated androstenedione to estrone and estradiol by aromatase enzyme in peripheral tissue. It acts by competitive binding to the haem of cytochrome P450 subunit of the aromatase enzyme and interrupts the metabolism of oestrogen resulting in a decrease level of oestrogen in the body. As a negative feedback mechanism, the pituitary gland produces follicle stimulating hormone (FSH) leading to ovulation or superovulation. Therefore Letrozole mimics the action of CC but without depletion of oestrogen receptors.¹⁸ It possibly avoids the anti-oestrogenic effects, associated with clomiphene citrate.¹⁸ Their half life in plasma is around 2 days.

Dose

Doses range from 5-7.5mgs, usually in two divided doses, by oral route. Should be taken daily for 5 days beginning on 2^nd/3^rd day of the cycle through 6^th/7^th day of cycle. Can also be started on 4^th or 5^th day of cycle and continued for a total of 5 days. ¹⁹

Side effects of Letrozole

Generally well tolerated, the most common adverse effects are hot flushes, nausea and hair thinning.²⁹ A greater number of new diagnosis of osteoporosis occurred among women receiving letrozole, but the long term effects on bone mineral density or lipid metabolism are yet to be determined.

Effectiveness of letrozole

Monthly pregnancy success rate with the use of letrozole ovulation induction/IUI is 5%-12% per cycle. Multiple birth rates is about 10% with the majority being twins.¹⁹

Potential advantages of Aromatase inhibitor for Ovulation induction¹⁸

High pregnancy rate.

· Monofollicular ovulation in most anovulatory patients.

· High safety due to short half life and few adverse effects.

· Reduced rate of multiple pregnancy

· Reduced risk of severe

· Ovarian Hyperstimulation syndrome

· Reduced FSH dose required for controlled ovarian stimulation

· Improved response to FSH in poor responders

· Low cost of treatment

· Convenience of administration: oral route, different regimens, including single-dose regimen

Induction of Ovulation with Human Gonadotrophins:

For over 30 years, the only treatment used for gonadotrophins was human menopausal gonadotrophins, a gonadotrophin extracted from urine of menopausal women. A commercial preparation is still available, with either 75 units of FSH and 75 units of LH per ampoule or in an ampoule with twice the amount, 150 units of each gonadotrophin. Gonadotrophins are orally inactive, so must be given parenterally; the heavy protein content of the urinary preparation requires intramuscular injections.

A more purified urinary FSH preparation became available by removing most of the LH in the urinary product. This product still requires intramuscular injection. A more highly purified form is available which can be administered subcutaneously.

Recombinant FSH is now produced in Chinese hamster ovary cells transfected with the human FSH subunit genes.¹²

Recombinant FSH is homogenous and free of contamination by proteins (characteristic of menopausal gonadotrophins from urinary extracts); this allows subcutaneous administration.

Because of its structural and biologic similiarity to LH, HCG, readily available from human pregnancy urine and placental tissue, is used to stimulate the mid-cycle LH ovulatory search.

Recombinant HCG and recombinant LH are now available. These preparations will allow patient self-administration by subcutaneous injections. It is unlikely that the results will be different; however, it is possible that the risk of ovarian hyperstimulation will be reduced.

· Preparation

· Human menopausal gonadotrophins

· Purified urinary FSH

· Highly purified urinary FSH

· Recombinant FSH

· Human chorionic gonadotrophins

· Recombinant HCG

· Recombinant LH

· Selection of patients for gonadotrophin treatment:

Demonstration of ovarian competence, tubal patency and uterine pathology should be evaluated, anovulation documented, and semen analysis obtained. Nongynaecological endocrine problems must be treated, hypogonadotrophic function, galactorrhoea syndrome must be evaluated for intra-cranial lesion.¹²

How to use gonadotrophin therapy:

A variable dosage method is used to achieve follicular growth and maturation. Follicle stimulation is achieved by 7-14 days of continuous gonadotrophins, one ampoule daily. Response is assessed by degree of oestrogen produced by growing follicles. The patients were monitored periodically with the measurement of the circulating estradiol level and vaginal ultrasound assessment of the number and size of follicles. The patient is seen on the seventh day of treatment, and a decision is made to continue or increase the dose (step up method). After the seventh day, the patient is seen anywhere from daily to every one or two days. Another approach, the step down method, starts with a higher doe (2-3 ampoules) and reduces the dose to one ampoule after initial response, theoretically approximating the changes in FSH in a normal ovulatory cycle.

Patients with polycystic ovaries are handled more gently. The increased sensitivity to FSH is apparently due to the availability of a larger cohort of small follicles ready to respond to FSH (recruitable follicles). In these patients, assessment starts on the fourth or fifth day. Excessive stimulation in women with polycystic ovaries can be avoided by lower doses of gonadotrophins extended over a longer duration of treatment.

When estradiol and ultrasound monitoring indicate that the patient is ready to receive ovulatory stimulus, 5000-10,000 units of HCG are given as a single dose intramuscularly. The patient is advised to have intercourse the day of the HCG injection, and two days afterward. In extremely hyposensitive ovaries, adequate follicular stimulation is required in doses of upto 4-6 ampules/day.

Pregnancy is usually achieved by administering gonadotrophins for 7-12 days.

Close supervision and experience is necessary to avoid difficulties.

Oestrogen Monitoring:

The use of oestrogen measurements is necessary to choose the correct moment for administering the ovulatory dose of HCG in order to prevent hyperstimulation. Estradiol measurement starts on day 7 of cycle.

An estradiol window of 1000-1500 pg/mL is optimal. The risk of hyperstimulatio0n is significant from 1500-2000 pg/mL and, as a general rule, over 2000pg/mL. HCG should not be given, and the ovarian follicles should be allowed to regress.¹²

Ultrasound Monitoring ³⁰

During normal cycles, the growing cohort of follicles can be identified by ultrasonography on days 5 to 7 as small sonolucent rays. The dominant follicle will become apparent by 8-10 days. Mean diameter of the follicle, indicating ovum maturity, varies from 20-24 mm (range 14-28 mm) in normal, spontaneous cycles. Pregnancies have not been observed with follicles <17mm in normal cycles. In 5-11% of cycles, two dominant follicles develop.

The potential signs of impending ovulation are: ³¹

· Presence of a dominant follicle (usually >16 to 18mm)

· Anechoic area, double contour, around the follicle

· Separation and folding of the follicle lining

· Thickened proliferative endometrium

Fluid can be detected by frequently, but not always, the cul-de-sac. The follicle disappears, or reappears as a smaller, irregular cyst, which diminishes in 4-5 days.

It is claimed that a higher pregnancy rate can be achieved when ultrasound is combined with oestrogen monitoring. The guiding principle has been to administer HCG when mature follicles correlate with an oestrogen level of 200-400 pg/ mature follicle (>13 mm).This principle only applies to several leading follicles. We believe that HCG should not be administered if there are more than 3-5 follicles 13mm or greater in diameter. Mild hyperstimulaiton has been associated with an increased number of intermediate size follicles.

Measurement of Endometrial Thickness³²

Studies in IVF cycles have revealed that successful implantation is correlated with endometrial thickness on the day of HCG administration. No pregnancies occurred when endometrial thickness was <6 mm. Chance of pregnancy is greatest if endometrial thickness is 9-10 mm or more.

Results with Gonadotrophin treatment

A cumulative conception rate of 40% after 6 treatment cycles can be achieved in women with unexplained infertility.In randomized, controlled clinical trials the monthly pregnancy rates in couples with unexplained infertility is increased 2 to 3 fold (monthly fecundity rate of 9%) with clomiphene treatment and with human gonadotrophins the monthly fecundity rate is 10-15% ³³ The risk of ectopic pregnancy increases with ovulation induction, as a consequence of multiple oocytes and high hormone levels.³⁴

Women with polycystic ovaries and moderate obesity require larger doses of gonadotrophins as compared to leaner women. A comparable pregnancy rate (40%) is achieved. However, spontaneous miscarriage is more frequent.

Complications:

· Hyperstimulation syndrome rate is 1-2 %.

· Multiple pregnancies

· Ovarian Hyperstimulation Syndrome (OHSS)

The incidence of clinically important hyperstimulation is striking, but mild type would be relatively common. The moderate to severe form appears at an impressive rate (1-2%).³⁵ Higher incidence of hyperstimulation is seen in IVF protocols, using combination of gonadotrophins and GnRH agonist.

Multiple pregnancies³⁶

Multiple pregnancy rates were formerly reported as approximately 20-30% (triplets or more 5%). Currently, it can be as low as 10-20% with careful monitoring and good medical judgement. Spontaneous twins occur at only about 1%, and triplets 0.010-0.017%. Dizygotic twinning rate is inherited from the mother. Monozygotic twins occur at a rate of 0.03-0.04%. In addition,miscarriage rate with gonadotrophins is higher (25%), probably due to effects of age, multiple pregnancies, and recognitions of early miscarriage.

Recent Studies on IUI and controlled ovarian hyperstimulation

Mitwally and Casper have shown in their study that- Letrozole is more effective than Clomiphene citrate either alone or in combination with Gonadotrophins for ovulation induction or augmentation.¹⁹ Patients treated with Letrozole demonstrate low oestrogen production per follicle, around the physiological level with good follicular development, and with a lower incidence of multiple pregnancy.¹⁸ When combined with Gonadotrophins, Letrozole reduces the dose of Gonadotrophins required for optimal follicular recruitment and improves the response to Gonadotrophin stimulation required in poor responders.¹⁹ Compared to Clomiphene citrate groups, in Gonadotrophin combined uterine insemination cycles, the number of mature follicles and serum estradiol level on the day of hCG- were significantly lower in the Letrozole group, but the pregnancy rate is higher in the latter.³⁷

In a randomized controlled trial in couples with unexplained non-conception, controlled ovarian stimulation (COS) in combination with IUI has been shown to result in significantly higher Cumulative pregnancy rate (PR) couple (33%) as compared to un stimulated intra Cervical insemination (10%), COS alone (19%) or IUI alone (18%)³³. The main benefit of the addition of COS to IUI is considered to be – induction of multifollicular growth (Cohlen, 1998)³⁸. In a study by M.M.E. van Rumste et al, (2006)³⁹ 32% of the couples conceived in four treatment cycles of COS- IUI. The pregnancy rate (PR per cycle in that study was 10.9%³⁹. Two European societies for human reproduction and embryology (ESHRE) multicentre prospective studies compared ovulation induction alone with ovulation induction in conjunction with IUI, Intra peritoneal insemination (IPI), GIFT³⁶. In the treatment of unexplained infertility, the pregnancy rate achieved from-superovulation alone was less than when combined with IUI³⁶, In a randomized controlled trial by F.P Hohmann and others(2001) low dose(75iu/dayof rFSH) initiated during early, mid and late follicular phase-led to multiple dominant follicle development.(overall in 60% cases) ⁴⁰ Martinez et al in an extensive review. The English Literature from 1980 to 1999 showed that there was a marked variation in the result of IUI between different clinics.⁴¹ Retrospective analyses of IUI data using life table analysis showed a relatively constant probability of becoming pregnant after each IUI treatment through four Gonadotrophin cycles.⁴² In a study of Fertility Institute of New Orleans (1983-98)-has been shown that the probability of pregnancy per cycle-depends on age, sperm, quality, sperm source and number of preovulatory follicles that developed in response to Clomiphene citrate (CC), ⁴³ and human menopausal Gonadotrophin (hMG) or follicle stimulating hormone (rFHS).⁴⁴ Data from Bourn Hall Clinic (2007) showed a clinical pregnancy rate of 22% per stimulated IUI cycle.

IUI is one of the most popular treatments in our country. Mild to moderate male factor subfertility can be overcome by controlled ovarian stimulation and intra uterine insemination. Although there are large number of study on IUI and COS internationally, but in our country there is no study at all.

The present study will be conducted to determine the follicular response by different types of ovulation inducing drugs in <30 and >30 age group of females, the conception rate in IUI in a tertiary level infertility management centre in our country.

Cost effectiveness of IUI and Ovulation induction

In a large retrospective analysis of 45 published reports on the treatment of couples with unexplained infertility, Guzick et al.⁴⁵ looked at the cost effectiveness of no treatment, CC alone, CC+IUI, hMG alone, hMG+IUI, IVF, and GIFT [26]. Analysis of data showed that CC+IUI was most cost effective (US$10,000 per pregnancy), compared to hMG+IUI (US$17,000 per pregnancy) and IVF (US$50,000 per pregnancy). In their conclusions, they state that “on the highest level of evidence found in the data” their recommendations of cost effective treatment for idiopathic infertility are: IUI does not seem to be effective without some form of superovulation, CC+IUI appears to be more cost effective than hMG+IUI; IVF and GIFT are effective for couples who have not conceived after superovulation +IUI.

In a study in Netherlands by Goverde et al., ³⁴ 258 couples with a diagnosis of idiopathic or mild male-factor infertility were divided into three equal groups: 1) IUI alone, 2) IUI with mild ovarian hyperstimulation using low-dose FSH, and 3) IVF [27]. Their conclusion was that stimulated IUI was as effective as IVF in achieving a pregnancy (31% vs. 33%), and was more cost-effective than IVF- cost per pregnancy resulting in a single live birth was US $4,511-5,710 for stimulated IUI, versus US$ 14, 679 for IVF. They concluded that patients should be counselled that IUI for these two diagnostic groups offers as good a chance of achieving a pregnancy as IVF and is more cost-effective.

Materials and methods

Methodology:

Study Design- prospective study

Place of study- The study was carried out in Centre for Assisted Reproduction (CARE), BIRDEM, Dhaka.

Duration of study- Starting from 10^th May 2010 to 31^st December 2010

Study Population- All infertility patients who attended CARE OPD BIRDEM

and selected for IUI during the study period was included in the study.

Sample Size- Sample size was determined using the following formula

n=z²pq/d²

Here, n=Desired sample size

z=Standard normal deviate usually set at 1.96.

q=1-p

d= degree of accuracy is considered as .05.

Prevalence of infertility 15%

p= .15

So sample n =

= 195.92

= 196

So the estimated sample size was 196 in one year. But for the convenience and time constraint all the patients of outpatient department of CARE BIRDEM who underwent IUI during the period of 10^th May 2010 – 31^st December 2010 was included in this study.

Total number of subjects, n=77.

Sampling technique:

Purposive Sampling Method. All the OPD patients who were selected for IUI in CARE BIRDEM during this defined period were included.

Written informed consent was taken from the patients. Then a detailed history, physical examination and base line investigations were done in all infertile couples who attended the OPD, CARE BIRDEM. Subjects were selected by the following inclusion and exclusion criteria.

Inclusion criteria:

Age of female < 40 years.

History of infertility at least > 2 years.

Unexplained infertility.

Mild to moderate male factor sub fertility with –

Total count >10 million / ml

Motility grade A > 10%

Morphology >30% N

Polycystic ovary syndrome. {resistant to clomiphene}

Mild endometriosis. {resistant to clomiphene}

Male factor – Erectile dysfunction, hypospadias

Exclusion criteria:

Bilateral tubal block.

Male factor subfertility where sperm count is <10 million / ml.

Age of the female partner >40 years.

Severe endometriosis.

No ovarian reserve.

Operational definition:

Infertility: Inability to conceive after 1 year of regular unprotected coitus.

Primary infertility: When the couple has no history of conception.

Secondary Infertility: When they have been successful on at least one previous occasion (Henry G et al 1997).

Fecundability: Probability of achieving pregnancy within one menstrual cycle.

Fecundity: Ability to achieve a live birth after being exposed to the risk of pregnancy for one menstrual cycle (Crosignani et al 1993).

Unexplained infertility Term applied to a subfertile couple whose standard investigations yields normal results.

Chemical Pregnancy: A pregnancy diagnosed by positive Beta hCG titre that spontaneously aborts before clinical verification by other means such as TVS.

Clinical Pregnancy: Pregnancy diagnosed by positive Beta hCG titre and clinically verified by TVS (intra uterine sac or foetal cardiac activity) or in case of a miscarriage by pathologic examination.

Intra Uterine Insemination (IUI): It is the reproductive technique where processed and prepared semen is directly introduced into the uterine cavity by a specialized catheter.

Controlled ovarian hyperstimulation and IUI (COH-IUI): Induction of ovulation is done by clomiphene or letrozole or low-dose gonadotrophins, where monitoring of follicular growth is done by Trans vaginal sonography, followed by IUI.

· Oligozoospermia (Oligospermia): Sperm concentration <20 million/mL

· Asthenozoospermia: Total motility<25% or forward progression<50.

· Teratozoospermia: Normal morphology <50% (or <15% Kruger strict criteria).

· Oligoasthenoteratozoospermia: Significant abnormalities of sperm.

· Concentration, motility, and morphology.

· Azoospermia: No spermatozoa in the ejaculate.

· Aspermia: No ejaculate.

Variables of the study:

Independent variable:

Type and dose of ovulation inducing agent

Dependent variable:

· Follicular size

· Endometrial thickness

· Research instrument: A structured questionnaire was given to the respondent.

Study Procedure: Eligible infertile couples were selected. Informed consent was taken from them. Almost all the patients had some sort of past infertility treatments from various parts of the country. So categorization of these patients was very difficult. Thus there were no fixed selection criteria to determine which group of patient will be in letrozole group and which will be in combined letrozole and gonadotrophin group. Thus the patients were divided according to age.

Group A:

Female with young age < 30 years.

Group B:

Female with age > 30, up to 40 years.

Superovulation Protocol:

Both groups of patients were stimulated by Letrozole 7.5 mg daily, started on D2 of the cycle and continued for 5 days. Patients were followed by this treatment for 3 cycles of IUI. Follicular response was assessed by TVS starting on D10 up to D14. When at least two follicles attained >18 mm size or single follicle >21 mm size then it was considered satisfactory. The patients who did not respond satisfactorily by 3 cycles of Letrozole were selected for combination of Letrozole and Gonadotrophins. These patients were stimulated by Inj. Gonadotrophins (rFSH) 50 iu intra muscularly daily starting from D3 for 5-7 days (or more if required) + Letrozole 7.5 mg per day on D2 – D6. Folliculometry was done by TVS starting from D10 upto D14 (depending on response). If at least 2 follicle size > 18 mm or 1 follicle is >21 mm, Inj. rFSH was discontinued and Inj. HCG (pregnyl) 5000 IUI was given intra muscularly. A single insemination was performed 36 hours after the Inj. of hCG (pregnil or professy 5000 iu).

Intra uterine insemination Protocol:

Semen specimens were collected by masturbation on site, evaluated according to standardized methods and prepared for IUI within 1 hr after collection. Semen was diluted 1:2 with HEPES buffered Ham’s F10 medium containing 1.5% serum albumin after centrifugation at 250 g for 10 min. The pillets were resuspended and combined in 3ml of medium and incubated for 1 hr. Appr. 0.05 ml was used to determine the concentration and motility of the sperm. 0.2-0.4 ml from the rest was drawn into the Shepard catheter, which is attached to 1 ml syringe. Then IUI was performed within 2.5 hrs of collection

Follow up procedure:

Patients were asked to attend the OPD of CARE BIRDEM 15 days after the missed period. Positive pregnancy test result was defined as pregnancy. Pregnant patients were advised to come for regular antenatal checkup.

Ovulation induction and IUI were offered to the patients who did not conceive, for the next two cycles.

Main outcome measure:

· Number of the follicle 2-3

· Size of the follicle >18 mm (if 2 or more), >21mm(1 or more) was considered satisfactory.

· Endometrial thickness >7 mm.

Data Collection:

Data was collected using a data collection sheet. Data from followup was aloso included in the data collection sheet. Follicular response was assessed by TVS report (D10 – D14) which showed number of the follicle, size of the follicle and endometrial thickness.

Data processing & analysis:

Result of the study was calculated & analyzed by standard statistical methods by using SPSS (Statistical Package for Social Science) software, version 16 (SPSS, Inc. Chicago, IL, USA) and Excel windows programme. The data was expressed as mean±SD and median (range) as appropriate. Student’s unpaired t-test and Mann-whitney u test was used for calculating the difference between the groups. ?² (chi square) test was performed to determine the clustering of different variable of interest. P value of less than 0.05 was considered statistically significant.

Ethical Consideration:

It was a prospective study; all the subjects selected for the study were informed orally and in written about the study design, objectives, and the right for the participant to withdraw from the project at any time for any reason. Written consent was obtained from each subject. Then approval from the Ethical review board BIRDEM was obtained.

Observation and Results

Fig 1

37 (48%) patients were in the <30 age group, and 40 (52%) patients were in the >30 age group.

Fig 2

15 (19%) suffered from infertility for <5 yrs, 30 patients (39%) suffered for 5-10 yrs, and 32 (42%) patients for >10 yrs. The mean duration of infertility was 9.4±4.7 yrs.

Fig 3

35 (45%) pts had normal FSH: LH ratio and 42 (55%) had an altered ratio. Hyperprolactinaemia was present in 15 (20%) patients. 3 (4%) pts were hypothyroid

Fig 4

.Polycystic ovaries were present in 42 (55%) patients. Endometrial thickness was <6 mm in 50 (65%) patients. Endometriosis was present in 9 (12%) patients, while 6 (7%) patients had fibroid uterus

Table 1

Patients Characteristics

Values	Mean	SD	Percentage (%)
Age	31.08	±4.86
Duration of infertility	9.45	±4.59
Parity
Nulliparous			77.92% (60 pts.)
Multiparous			22.8% (17 pts.)
Clinical Presentation
Oligo/anovulation			74.02% (57 pts.)
Hyperandrogenism			14.28 % (11 pts.)
PCOS			55.05% (42 pts.)
BMI (weight in kg/m2)	22.7	±3.24

Mean age of the patients were 31.08 ±4.86 yrs. Mean duration of infertility is 9.45 ±4.59 yrs. Mean BMI was 22.7±3.24.

Table 2

Effect of letrozole and combined letrozole and gonadotrophins on follicular size

Variables	Letrozole Group(n=77)	Combined Letrozole and Gonadotrophin Group (n=65)
No follicles	38 pts (49.36%)	Nil
Pts with follicle size 10-14mm	14 pts (18.9 %)	Nil
Pts with follicle size 14-18mm	13 pts (16.88 %)	15 pts (23.07%)
Pts with follicle size >18mm	12 pts (15.58 %)	50 pts (76.93%)

Variables	Letrozole (n=77)	Combined Letrozole and Gonadotrophin (n=65)	t	?2	P
Total no. of follicles	1.28±0.94	3.37±0.87	7.54		0.0001
No. of mature follicles (>18mm)	(12 pts)3.54±1.63	(50 pts)3.44±1.93	0.374		0.71
Pre-treatment endometrial thickness	5.93±1.06	5.93±1.07	5.37		0.67
Post-treatment endometrial thickness	7.14±1.70	8.61±1.4	5.83		0.0001
Duration of stimulation (days)	12.2±1.3	10.3±2.8
Ovulation/cycle	43/231 (18.6%)	174/195 (89.2%)		208.17	0.0001

In letrozole group, mature follicles (>18 mm) developed in only 12 (15.58%) patients.

On the contrary, by giving gonadotrophins along with letrozole, mature follicles appeared in 50 (76.93%) patients.

Table 3

Outcome in letrozole and combined letrozole and gonadotrophins

The mean of total no. of follicles in letrozole group is 1.28±0.94, whereas in combined letrozole and gonadotrophins group, the mean is 3.37±0.87.

Average duration of stimulation was 12.2±1.3 days for letrozole group, and 10.3±2.8 days for combined letrozole and gonadotrophins group. Ovulation/cycle was 18.6% in letrozole group and 89.2% in combined letrozole and gonadotrophins group

Table 4

Endometrial thickness in letrozole and combined letrozole and gonadotrophin

Variables	Letrozole (n=77) (cycle=231)	Combined Letrozole and Gonadotrophin (n=65) (cycle=195)	t	?2	p value
Endometrial Thickness >7mm	37 pts (48%)	58 pts (90%)		25.16	0.0001
Endometrial Thickness <7mm	40 pts (52%)	7 pts (10%)		25.16	0.0001
Post-treatment endometrial thickness	7.14±1.70	8.61±1.4	5.83		0.0001

Endometrial thickness after treatment by letrozole was 7.14±1.70 mm, and post treatment endometrial thickness by combined letrozole and gonadotrophins was 8.61±1.4 mm, and p value was 0.0001, which is significant.

Table 5

Comparison of follicular response between two age groups (<30 yrs & >30 yrs) by letrozole

Induction used – Letrozole (7.5 mg/ d for 5 days in a cycle)

Variables	<30	>30	t test	?2	P
No. of pt	37	40
No. of induction	111	120
Follicular size >18mm	8 pts (22%)	4 pts (10%)		0.34	0.74
Endometrial thickness(mm)	8.52	8.7	0.556		0.58
Ovulatory cycles (%)	28/111 (25%)	15/120 (12.5%)		3.82	0.051
Pregnancy rate/cycle (%)	9/111 (08%)	3/120 (03%)		2.63	0.11
Pregnancy patient rate