Tejshree Singh, MBBS, CPSDGO, MD, DNB*, Abha Majumdar, MBBS, MS, FICS**
*Consultant Infertility Unit, Department of Obstetrics and Gynaecology, Kolmet Hospital & Research Centre, New Delhi, India
**Senior Consultant and Head of the Unit of IVF and Human Reproduction, Department of Obstetrics and Gynaecology
Subjects and Methods Results To compare the efficacy and tolerability of freeze-dried and liquid formulations of urinary human chorionic gonadotropin (hCG) for inducing final oocyte maturation and triggering ovulation.
In a randomized, single-centre study, infertile women (n=100) received single injections of urinary hCG (5000 IU) from reconstituted freeze-dried (1.0 mL of 5000 IU /mL) and liquid (0.5 mL of 5000 IU /0.5 mL) formulations in random order, when their ultrasounds showed one or more dominant follicles of size 18mm or above. Blood samples were taken after 36h for determination of hCG concentration by immunoassay using direct chemilluminometric technology. Follicular rupture was assessed by means of another ultrasound undertaken 48h following the injection of hCG and serum progesterone levels undertaken 8 days later
The mean serum hCG levels achieved 36 hours post hCG injection were 74.05 mIU/ml for the freeze-dried formulation and 69.65 mIU/ml for the liquid formulation. The mean difference between these levels was 4.400 (95%confidence interval -19.002 to 10.202) which is statistically not significant (p-value 0.545). These levels were equally effective for triggering ovulation. The mean serum progesterone levels achieved day 8 post hCG, were 8.452 ng/ml for the freeze-dried formulation and 8.228 ng/ml for the liquid formulation. Both formulations were tolerated well; the liquid formulation of hCG was associated with fewer adverse effects.
The levels of hCG required to trigger follicular rupture are anywhere between 20 to 45 mIU/ml. As ovulation will happen about 36-48 hours after the injection of hCG, procedures can be scheduled to take advantage of this time sequence.
The liquid formulation of recombinant hCG was shown to be bioequivalent to the freeze-dried formulation, with the same efficacy and no clinically significant differences in tolerability.
Our study shows that the liquid formulation of urinary hCG provides the same efficacy as the freeze-dried formulation when used to trigger final follicular rupture, is convenient to use and has better tolerability.
Preimplantation genetic screening for all 24 chromosomes by microarray comparative genomic hybridization significantly increases implantation rates and clinical pregnancy rates in patients undergoing in vitro fertilization with poor prognosis
ABSTRACT
CONTEXT: A majority of human embryos produced in vitro are aneuploid, especially in couples undergoing in vitro fertilization (IVF) with poor prognosis. Preimplantation genetic screening (PGS) for all 24 chromosomes has the potential to select the most euploid embryos for transfer in such cases. AIM: To study the efficacy of PGS for all 24 chromosomes by microarray comparative genomic hybridization (array CGH) in Indian couples undergoing IVF cycles with poor prognosis. SETTINGS AND DESIGN: A retrospective, case – control study was undertaken in an institution based tertiary care IVF center to compare the clinical outcomes of twenty patients, who underwent 21 PGS cycles with poor prognosis, with 128 non PGS patients in the control group, with the same inclusion criterion as for the PGS group. MATERIALS AND METHODS: Single cells were obtained by laser assisted embryo biopsy from day 3 embryos and subsequently analyzed by array CGH for all 24 chromosomes. Once the array CGH results were available on the morning of day 5, only chromosomally normal embryos that had progressed to blastocyst stage were transferred. RESULTS: The implantation rate and clinical pregnancy rate (PR) per transfer were found to be significantly higher in the PGS group than in the control group (63.2% vs. 26.2%, P = 0.001 and 73.3% vs. 36.7%, P = 0.006, respectively), while the multiple PRs sharply declined from 31.9% to 9.1% in the PGS group. CONCLUSIONS: In this pilot study, we have shown that PGS by array CGH can improve the clinical outcome in patients undergoing IVF with poor prognosis
INTRODUCTION
The last decade has seen a focused and persistent effort toward developing means and technologies to identify embryos that are most likely to implant and grow till term resulting in the birth of a healthy live baby. At present, morphology evaluation is the mainstay of embryo selection since it is noninvasive and easy to perform. However, it has not proved to be a very efficient method for selecting embryos since implantation rates and clinical pregnancy rates (PR) per transferred embryo continue to be very low. Among many factors, one major reason for poor reproductive potential of embryos generated in vitro is the prevalence of aneuploidies in such embryos. The rate of aneuploidy rises with increasing maternal age] but it is only moderately associated with morphology. Therefore, a significant percentage of even the “most ideal†embryos selected for transfer will be aneuploid, resulting in poor reproductive outcome. Moreover, transferring aneuploid embryos can be potentially dangerous since aneuploidy is the most common cause of miscarriage and the most common genetic abnormality in embryos. Preimplantation genetic screening (PGS), even though highly invasive in nature, started out as a very promising concept allowing embryos to be screened for aneuploidies before being selected for transfer. However, this older version of PGS failed to live up to expectations, as numerous authors failed to show improvement in in vitro fertilization (IVF) outcomes with PGS using fluorescence in situ hybridization (FISH), in which chromosomal analysis of not all but only a few chromosomes was performed. With the advent of new validated platforms for comprehensive chromosomal screening (CCS) such as single nucleotide polymorphism array, microarray comparative genomic hybridization (array CGH), and quantitative polymerase chain reaction (PCR), capable of analyzing all 24 chromosomes, now an improved version of PGS involving 24 chromosome copy number analysis is being expected to be a likely remedy for the earlier shortcomings. This retrospective case–control study seeks to examine the efficacy of PGS applied to poor prognosis patients, given the relative paucity of information concerning the use of modern 24 chromosome copy number analysis for this patient group. The objectives of this study were to establish the incidence of aneuploidy in such patients undergoing IVF with poor prognosis and to undertake a retrospective comparative analysis of the clinical outcomes of these patients undergoing IVF PGS cycles with non PGS, IVF controls.
ABSTRACT
OBJECTIVE: To study the prevalence of metabolic syndrome (MBS) in Indian women and to see how does it correlate to body mass index (BMI) and polycystic ovarian syndrome (PCOS) in this population. STUDY DESIGN: Prospective cross sectional observational study. SETTING: Infertility clinic of a tertiary center. MATERIALS AND METHODS: Two hundred women, 120 with PCOs and 80 age-matched controls were enrolled. The prevalence of MBS was studied in the women with and without and was co related to BMI by further subgrouping as team (BMI 23 kg/m2). The sample size was: team controls-40, obese controls-40, team PCOS80. Each subject underwent a physical examination and laboratory evaluation for the diagnosis of MBS, which was defined according to the guidelines of National Cholesterol Education Program Adult Treatment Pamel (NCEP ATP III) 2005. INTERVENTION: None. MAIN OUTCOME MEASURES: Main Outcome Measures: Subjects with and without PCOs were compared with each other for the prevalence of MBS, and similarly team subjects were compared with obese subjects. Receiver operator characteristic (ROC) curves were obtained for both the PCOS and non PCOS population separately, co-relating the prevalence of MBS with BMI. These ROC curves were used to establish the cut off values of BMI, which could best predict the risk of MBS. RESULTS: The prevalence of MBS was significantly higher in the women with PCOS, as compared to age matched controls. Similarly, when BMI was considered, MBS was more prevalent in overweight subjects than in lean subjects with or without PCOS. In subgroup analysis, the presence of PCOS had a lesser impact on the prevalence of MBS as compared to non PCOS controls with higher BMI. The relative risk of MBS increased as follows: lean controls 1, lean PCOS 2.66, obese controls 5.33, and obese PCOS 6.5. The most appropriate cut off level of BMI for predicting the risk of MBS in Indian women without PCOS seems to be 23 kg/m2 , whereas, with PCOS, it was 22.5 kg/m2 . CONCLUSION: MBS is more prevalent in women with PCOS. However, obesity is an independent and stronger risk factor for developing MBS. To reduce the risk of MBS and its related long term health consequences, lifestyle modification is advisable above BMI of 23 kg/m2 in the normal population and 22.5 kg/m2 in women with PCOS.
INTRODUCTION
Polycystic ovarian syndrome (PCOS) is common disorder, affecting approximately 5-10% of the women in reproductive age group.[1,2] It is characterized by chronic anovulation, hyperandrogenism, and polycystic ovaries. The other metabolic abnormalities associated with PCOS are obesity, dyslipidemia, insulin resistance, glucose intolerance, and hypertension, which confer an increased risk of long term health consequences such as type II diabetes mellitus and cardiovascular risk.[3] Most of these metabolic features are also shared by the syndrome X or metabolic syndrome (MBS), which is associated with atherosclerosis, hypertension, dyslipidemia, coronary artery disease, and diabetes. Some of the factors affecting the prevalence of MBS are age,[4,5] obesity,[4] insulin resistance,[6] and underlying PCOS.[7a] Till date, there are few studies, mainly from the American population[8 10] and still fewer from the European continent,[11,12] which have addressed the prevalence of MBS in women with PCOS. There is a scarcity of data from Asian population.[13,14] Moreover, in most of these studies, the women with PCOS were found to have a higher body mass index (BMI) and waist circumference, as compared to the controls, which could have confounded in the higher prevalence of MBS. This study was undertaken with an aim of comparing the prevalence and different characteristics of MBS in Indian women with PCOS and age matched controls. To negate the confounding effect of the high BMI associated with PCOS, subgroups of lean and obese women were studied separately for the prevalence of MBS. In order to reduce the serious long term consequences related to MBS, we have attempted to find out the predictors of MBS, and the action points, at which screening for MBS and lifestyle modification would be beneficial, in respect to preventing, or modifying long term morbidity.
Over the last three and a half decades after the first successful in vitro fertilization (IVF), the technology has been rolling forward in all directions; so that a technique which was once considered the last resort to successful fertility has almost become the first choice. Why is it so? Is IVF flawless, without complications, and has the highest probability of fulfilling ones wish to correct the underlying disorder causing sub-fertility and promote natural conception appear redundant today! One would argue that it is not so; IVF has a flip side-complications, side effects, failures, and financial and emotional exhaustion are all part of it. But then, on the other hand, this technique is truly the only way to parenthood for a lot of medical conditions that cannot be rectified by developments within the scope of drugs and surgery. It is for this sector of patients who crave for a child, who are willing to forfeit the increased risk of congenital abnormality as well as maternal complications imposed by the technology in order to fulfill their dream of parenthood.
Originally, IVF was the only way out for women with blocked or irreparably damaged fallopian tubes. With the advent of intracytoplasmic sperm injection (ICSI), the indications of IVF expanded; initially to severe male factor infertility and then to couples with unexplained infertility. With the development of oocyte and embryo donation as well as surrogacy, the use of IVF could be extended to a lot more couples who would otherwise not have a chance to parent their own child. All these techniques involving use of third party for lending their gametes, embryos, or uteruses have made it possible for women with ovarian failure or premature menopause, couples with no gametes, and for women with absence of a functional uterus respectively to be able to carry a child. Becoming a gestational or true genetic mother has turned into reality for so many desperate mothers. Lately, the technology of oocyte and ovarian tissue freezing for fertility preservation before going through chemotherapy has become another turning point in the life cycle of IVF which now enables women to be able to parent their own child at a later date. Recently, the development of preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) by testing one cell from “in vitro embryos” has further increased the possibility for couples to have disease free healthy babies.
However, after having said all this about the use and applications of this rapidly advancing technology, we also must understand that human reproduction is a very inefficient process and IVF mirrors this “wastage” at every stage. One in five cycles undertaken to entangle many more desperate couples into IVF who have become old wanting a child and will refuse to see any health risks associated with pregnancies at this age.
The IVF industry is powered by photographs of beautiful babies held by proud parents. Being childless is so traumatic for manythat almost any cost seems worth paying. Indeed, the birth of a desperately wanted child is a priceless miracle for a couple who has otherwise given up all hope. The technology must continue to grow as it is one of the greatest advancement in the field of medicine where “life is created outside the human body”. All we need is good and strong legislation to safeguard mothers as well yields no eggs at all, all retrieved oocytes do not fertilize, and 90% of embryos replaced do not survive. Of those that do, a few result into life-threatening ectopic pregnancies requiring urgent abortion while others miscarry. The number of peri-natal deaths is also double the rate of spontaneous conceptions partly because multiple pregnancies are more common with IVF and often result in premature delivery.
More than a failure is the iatrogenic complications of this technology. Amongst the foremost complications of IVF is the dreaded ovarian hyper-stimulation syndrome (OHSS) which is the direct effect of drugs used for ovarian stimulation and may become a real life threatening hazard. One woman in 12/cycle has mild symptoms, but two in every 100 are so ill that they need hospital admission and intensive care on account of the complications of OHSS. However, the biggest price to be paid cannot just be measured in terms of money or health risks. It is the emotional roller-coaster which goes with drugs used for treatment, egg retrieval, semen collection, embryo replacement, freezing of surplus embryos, and above all, high hopes and crushing disappointment.
At this stage with so many advances happening globally came the role of the middle man who tried to bring a third party to the intending parents. Policymakers, who could foresee and perceive the antecedent exploitation of such developments, restricted the use of third-party reproduction in their jurisdiction and countries. However, a lot of developing countries gave into these newly developed technological advancements, partly because of lack of a regulatory governing body and mostly because of monetary gains. ICSI started being used for all women undergoing IVF, oocyte donation to compensate for sub-optimal standards of IVF clinics, embryo donation for anyone and everyone especially for women of over 50 years of age, surrogacy for social reasons as well as for single fathers and gay couples, oocyte freezing for women wanting to delay pregnancy for personal and professional reasons, and PGD and PGS for sex selection of the unborn baby.
Out of the so many enumerated “misuses” of the technology, there are instances where law has been trying to control the health of women by limiting the age of surrogates to 35 and the age of oocyte donors also to below 35 to maximize the genetic normalcy of the unborn child. Nevertheless, there is no law to limit the age
of a woman wanting to carry a child for her own self by donated oocyte or donated embryo. This presents yet another risk in that even if we were to set aside the exaggerated maternal mortality and morbidity with these older age pregnancies, we also seem to be unfairly deciding the fate of the unborn child by allowing them to be parented by couples who are aged like grandparents and are no longer capable of providing the same care a younger couple is able to provide, the way nature intended it. Who has given us the right to choose the destiny of these children? Or is this another “feather in our cap” which we very proudly advertise as the unborn child who might get entangled in this rut of human race unknowing and unaware of the complications.
INTRODUCTION
Ovulation is the result of a maturation process that occurs within the hypothalamic-pituitary-ovarian (HPO) axis and is orchestrated by a neuroendocrine cascade terminating in the ovaries. Any alteration in this results in a failure to release a mature ovum, leading to anovulatory cycles. Anovulatory disorders may manifest in a variety of clinical presentations, ranging from luteal insufficiency to amenorrhea. Anovulatory disorders account for about 30 to 40 percent of all cases of female infertility. These disorders are generally amongst the most easily diagnosed and most treatable cause of infertility.1 Most of these women have oligomenorrhea, arbitrarily defined as menstruation that occurs at intervals of 35 days to 6 months. While ovulation may occasionally occur, spontaneous conception is unlikely. Therapeutic options have expanded significantly in recent years from clomiphene to gonadotropins. Induction of ovulation in these women is aimed at inducing monofollicular development and subsequent ovulation. Induction of ovulation should be differentiated from stimulation of multiple follicle development in ovulatory women, as is done in association with assisted reproductive technologies. The method of ovulation induction (OI) selected by the clinician should be based upon the underlying cause of anovulation and the efficacy, costs, risks and potential complications associated with each method as they apply to the individual woman. This chapter reviews the common types of anovulation including their pathogenesis, diagnosis and efficacy of the different treatment regimens that are used and our approach to the management of such woman. Some of these disorders are reviewed in greater detail elsewhere in this book.
WORLD HEALTH ORGANIZATION CLASSIFICATION OF ANOVULATION
The clinical approach in management of patients with ovarian dysfunction requires an understanding of its causes. Classification of ovulatory dysfunction based on their cause was originally described by Lunenfeld and lnsler and then by European Society for Human Reproduction and Embryology (ESHRE) and again modified by World Health Organization (WHO). According to WHO, ovulatory dysfunction is classified into three main categories on the basis of menstrual cycle length; oligomenorrhea (menstrual cycles > 35 days) or amenorrhea (menstrual cycle > 6 months) in combination with serum hormonal levels of prolactin (PRL), follicle stimulating hormone (FSH) and estradiol (E2) (Box 1).
WHO CLASS 1
Hypogonadotropic Hypogonadism This group accounts for 5 to 10 percent of anovulation. Women in this group, frequently present with amenorrhea.
Etiology Hypogonadotropic hypogonadism (HH) can be congenital or acquired. Congenital HH is further divided into anosmic HH (Kallmann’s syndrome) and normosmic isolated HH (idiopathic HH). Kallmann’s syndrome itself is a heterogeneous disorder with an X-linked form due to mutations in Kal1 gene (the gene encoding for anosmin-1 protein) and other forms with autosomal transmission. Pathology of idiopathic HH is failure of gonadotropinreleasing hormone (GnRH) neurons in the hypothalamus to differentiate or develop resulting in either lack of or apulsatile GnRH secretion.
The world has witnessed many developments in the field of assisted reproduction since the birth of Louise Brown in 1978, a product of in-vitro fertilization. What has happened in the last thirty-four years or so is a story to be told: A story of realization, discoveries, execution, performance and triumph. Following closely in the heels of this first assisted reproductive technology, were the development and execution of other incredible techniques: freezing of gametes and embryo and their subsequent successful use for reproduction (1983, 1985), pre-implantation genetic diagnosis by embryo biopsy(1990), Intra-cytoplasmic sperm injection (1991), use of testicular sperms from azoospermic males to have them father their own genetic child and cryopreservation of ovarian and testicular tissue in cancer patients for future fertility preservation.
Louise’s birth was not a chance occurrence but a result of years of dedicated research. At the time, achieving an implantation rate of more than 2-3% per embryo transferred was unthinkable. Researchers set upon the task of improving this implantation and thereby the success rate, mainly by focussing on methods to increase oocyte recovery, better culture conditions, improving transfer techniques and luteal support.
Within 20 years, the implantation rates in various laboratories had crossed 15-20%. However, to have the highest pregnancy rates, 3 to 4 embryos were transferred in every IVF cycle. What this meant was that the chances of all embryos implanting and hence leading to multiple births also increased which greatly increased the associated risks of perinatal mortality and morbidity.
Therefore, the focus shifted towards developing methods to improve IVF success rates, while reducing twin and triplet pregnancies. The approach was to develop screening methods to identify the most viable embryo, so that transfer of fewer healthy embryos would result in a higher proportion of singleton pregnancies.
One method used was to prolong culture of embryos in vitro by optimizing culture conditions, so that natural selection of only the more viable embryos (blastocysts day 5 embryo) for transfer came to the fore.
Another way of knowing whether the embryo was robust enough to implant was by checking if it was genetically normal, and this was achieved by biopsying cell/s from the embryo. This cell was then subjected to cytogenetic techniques like FISH to determine aneuploidy; or to molecular techniques like PCR to determine a known point mutation on a particular chromosome.
A third way to pick up the most viable embryo was to check their metabolic health through what is known as “metabolomics”. This involves profiling each embryo based on the quality and quantity of their excretome, as well as by assessing the oxygen uptake by embryos.
What currently seems to be holding the world’s attention is the visual documentation of growth and cleavage-rates of embryos through an incubator-mounted, time lapse camera which is simply termed the “time lapse” or “dynamic morphometric analysis”.
While advances were being made within the embryology lab, exciting changes were also happening in the world of gonadotropins. The urinary gonadotropins were slowly giving way to the self administered, purified, recombinant gonadotropins. Better timing of oocyte retrieval, due to avoidance of pre-mature LH surge, could be attained with the use of GnRH analogues (agonists from 1983 onwards, and antagonists from 1999 onwards). Recently, to ease the pain of daily injections, long acting Gonadotropin injection (elonva), which need only be given once in seven days, has been developed. Also, work is underway to prepare an oral formulation of gonadotropins.
With better stimulation techniques, the ovarian response often over-stepped the desired mark of safety, leading to ovarian-hyper-stimulation syndrome, a life threatening iatrogenic condition. Advancements towards reverting to soft stimulation protocols to lessen the incidence of ovarian hyper-stimulation and yet obtain adequate number of oocytes and embryos to maintain good pregnancy rates are being made.
One of the many controversial areas of assisted reproduction has been cloning. Animal cloning has been achieved by the process of somatic cell nuclear transfer (1996). The same technique has been employed for cloning of human embryos as well (2009). The human fertilization and embryology authority prohibits cloning of human embryos for the purpose of reproduction since it deems to totally bypass nature’s law of sexual reproduction. Whether the first cloned human will ever be born is yet to be known.
For future generations, by working on a background of these achievements, one can only hope that their task of making assisted reproduction more efficient and complication-free, more available to a wider range of patients, and more socially and economically acceptable, would become easier.
OBJECTIVES:
To evaluate whether three daily doses of GnRH agonist (Inj. Lupride 1 mg SC) administered 6 days after oocyte retrieval increases ongoing pregnancy rates following embryo transfer (ET) in cycles stimulated with the long GnRH agonist protocol.
SETTINGS AND DESIGN:
Prospective randomized controlled study in a tertiary care center. MATERIALS AND METHODS: Four hundred and twenty six women undergoing ET following controlled ovarian stimulation with a long GnRH agonist protocol were included. In addition to routine luteal’ phase support (LPS) with progesterone, women were randomized to receive three 1 mg doses of Lupride 6 days after oocyte retrieval. Computer’ generated randomization was done on the day of ET. Ongoing pregnancy rate beyond 20th week of gestation was the primary outcome measure. The trial was powered to detect a 13% absolute increase from an assumed 27% ongoing pregnancy rate in the control group, with an alpha error level of 0.05 and a beta error level of 0.2.
RESULTS:
There were 59 (27.69%) ongoing pregnancies in the GnRHa group, and 56 (26.29%) in the control group (P = 0.827). Implantation, clinical pregnancy and multiple pregnancy rates were likewise similar in the GnRHa and placebo groups. CONCLUSIONS: Three 1 mg doses of Lupride administration 6 days after oocyte retrieval in the long protocol cycles does not result in an increase in ongoing pregnancy rates.
INTRODUCTION
Luteal “phase deficiency occurs frequently in ovulation induction cycles using pituitary down” regulation with a GnRH agonist as well as in those using GnRH antagonists for ART. To cope with this problem, different regimens of luteal’ phase support have been suggested. Most of these regimens involve use of two therapeutic agents, HCG and progesterone, used separately or together in different doses and routes of administration. Adjuvants have been tried to further better the luteal support with either of these two agents used primarily for luteal support such as estradiol, aspirin, vitamin C etc., Some recent data, however, have suggested a beneficial effect of GnRH agonist administered in the luteal phase on ART outcomes. The mechanism of the presumed beneficial effect of luteal’ phase GnRH agonist administration is not clear.
and may be due to the drug action at multiple levels. It was hypothesized that GnRH agonist may support the corpus luteum by stimulating the secretion of LH by pituitary gonadotroph cells or by acting directly on the endometrium through the locally expressed GnRH receptors or by directly acting on the embryo itself. The administration of a single dose of GnRH agonist in the luteal phase was also shown to increase pregnancy, implantation, delivery and birth rates in recipients of donated oocytes in whom ovulation was suppressed and the corpus luteum was thus absent, suggesting a direct effect of GnRH agonist on the endometrium or the embryo. Despite stressing the importance of this finding, properly conducted trials to evaluate the reproducibility of these results were advised before widespread adoption of this simple yet seemingly effective strategy. In this report, we present a prospective randomized, controlled trial aimed to assess the effect of a three 1 mg doses of Inj. Lupride, administered 6 days after oocyte retrieval, on the probability of ongoing pregnancy rates following embryo transfer (ET) in cycles stimulated with a long GnRH agonist protocol.
Summary:
Reporting two cases of primary amenorrhea with primary infertility and underlying hypogonadotropic hypogonadism (HH). In both cases ovulation induction was initiated with highly purified human menopausal gonadotropin (hMG) in doses starting from 75 IU to the highest of 450 IU per day for 34 and 23 days respectively, with no follicular response and steroidogenesis. In the same cycle the gonadotropin preparation was changed to recombinant follicle stimulating hormone (r-hFSH) and recombinant luteinizing hormone(r-hLH) in doses of 225IU and 75IU respectively in the first case for 9 days and 150IU and 75IU respectively for 6 days in the second case, after which dominant follicles developed with subsequent ovulation. Both treatment cycles resulted in term live births. Hence, it appears that recombinant gonadotropins are more consistent in their activity as compared to the available urinary gonadotropins.
Background:
Hypogonadotropic hypogonadism is a condition of failure of gonadal function, characterized by low or undetectable levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) with subsequent estradiol deficiency and primary amenorrhea. The gonadotropin deficiency is owing to absence of pulsatile secretion of hypothalamic GnRH, or pathology within or around the pituitary which leads to complete arrest of ovarian follicular growth.
Pregnancy can be achieved in this condition through ovulation induction with exogenous gonadotropins. Gonadotropins were developed for clinical use for the first time in 1957, from urine of menopausal women. However, there were multi faceted problems in using gonadotropins recovered from this source. These problems ranged from collection of large quantities of urine to presence of urinary proteins in purified extracts, low specific activity causing inconsistency in the amount of gonadotropin, along with batch to batch variability. Ever since this development, scientists have been on the lookout of a more consistent preparation of gonadotropins which could take care of all these deficiencies. Recombinant gonadotropins were developed by the recombinant DNA technology and launched first time for clinical use in 1995 after approval by the European Medicines Evaluation Agency. These gonadotropins had batch to batch consistency with minimal variability in drug effect and high specific activity.
We are presenting here two cases of HH, who were started for ovulation induction with urinary hMG with almost no follicular response even after 34 and 23 days of administration respectively. Surprisingly, on changing the type of gonadotropin further to recombinant FSH and recombinant LH during the same cycle, follicular response was achieved within 9 and 6 days in both cases respectively, to cause successful ovulation and subsequent live birth.
Case presentation:
Case-1 A 30 years old female with primary amenorrhea came with secondary infertility. She had one miscarriage which she conceived through ovarian stimulation and intrauterine insemination. Hormonal assay diagnosed her as a case of hypogonadotrophic hypogonadism. Injections of hMG were given starting from 75 IU/day in incremental doses, up to 450 IU/day for duration of 34 days with no recruitment of follicles. Subsequently r-hFSH and r-hLH were used in 3:1 ratio (225:75 IU) replacing hMG following which ovulation of two dominant follicles was achieved with 9 days of stimulation.
Case-2 Our second case was a 32 year old hypogonadotrophic hypogonadic woman with primary amenorrhea and primary infertility. Ovulation induction was initiated with 75 IU/day of hMG and the dosage was increased to 450 IU with no recruitment of follicles till 23 days of drug administration. We switched over to r-hFSH and recombinant r-hLH in 2:1 ratio (150:75 IU) and administered these for 6 days on a daily basis after which 2 dominant follicles were obtained, which ovulated following human chorionic gonadotropin (hCG) trigger.
Investigations:
|
|
Case-1 |
Case-2 |
|
AMH(pMol/L) |
10.2 |
9.4 |
|
FSH(mIU/ml) |
0.3 |
0.41 |
|
LH(mIU/ml) |
0.1 |
0.72 |
|
Estradiol(pg/ml) |
10 |
20 |
|
Pre-hCG estradiol(pg/ml) |
329 |
137 |
Differential diagnosis:
Ovarian dysgenesis (OD) is the most common differential diagnosis due to similar clinical presentation of primary amenorrhea, delayed puberty and infertility.
Serum gonadotropin levels are most significant in order to differentiate between the two. OD is characterized by high FSH levels with irregular release, less elevated and more stable LH level, while HH reveals sharply reduced monotonous FSH and LH levels.
Outcome:
In both patients, 2 dominant follicles were formed after change over to r-hFSH and r-hLH. Pre-ovulatory estradiol levels were 329pg/ml and 137pg/ml in case 1 and 2 respectively. Both of them conceived with twin pregnancy and subsequent live births at term gestation.
Discussion:
In normo-gonadotropic females basal FSH and LH is sufficient for ovarian response, hence, stimulation with FSH alone leads to optimal folliculogenesis with adequate steroid production. However, in HH the deficiency of FSH and LH both is profound and as per “two cells two gonadotropin” theory both the gonadotropins are essential for adequate follicular growth and steroidogenesis. Pulsatile GnRH was the first method used for gonadotropin secretion leading to monofollicular ovulation in such patients. Later on pulsatile GnRH agonist was abandoned in favor of hMG due to need for the cumbersome electronic pump, inconvenience of drug administration as well as the side effects and complications of using pulsatile GnRH. Therefore, hMG became the mainstay of treatment as it contained both FSH and LH in 1:1 ratio hence, could provide both the essential gonadotropins in such cases. However, urinary gonadotropins are considered to be inconsistent in their action with variable and low specific activity as compared to recombinant human gonadotropin, which apart from having batch to batch consistency also has very high specific activity. This possibly appears to be the reason of the beneficial effect seen in follicular growth on changing over from hMG to recombinant gonadotropin in both our cases. A recent systematic review and meta-analysis compared the efficacy of urinary gonadotropin to recombinant gonadotropin in intrauterine insemination cycles for women with unexplained infertility and concluded that recombinant preparation was required in half the dose when compared to highly purified urinary preparation to achieve the same pregnancy rate. However, when both gonadotropin where used in the same dose pregnancy rates was significantly lower in the urinary gonadotropin group. Another recent study from Italy compared recombinant gonadotropin to hMG in 70 women with HH and found similar ovulation rates in both groups but significantly higher pregnancy rates in the recombinant gonadotropin group.
Learning points:
Administration of recombinant gonadotropins has shown in most trials to have more consistent action with no batch to batch variability. Addition of r-hLH in minimal doses to r-hFSH appears optimal in terms of folliculogenesis and steroid production to achieve ovulatory response even in cases of extreme gonadotropin deficiency such as in hypogonadotropic hypogonadism.
Introduction
Nearly one-third of the world population is infected with Mycobacterium tuberculosis (MTB) of whom only 10% are known to progress to clinical disease (WHO, 2006). Depending upon the localization of the MTB in an organ, a wide spectrum of tubercular disease is encountered in clinical practice of which female genital TB (GTB) is an important manifestation (Schaefer, 1976; Parikh et al., 1997; Aliyu et al., 2004; Dannenberg and Converse, 2011). Damage to the pelvic organs after clinical GTB is well recognized both in the presence of active disease as well as during the process of healing and fibrosis. The diagnosis can be established in such cases with the help of various microbiologic, radiologic and histopathologic tests along with the clinical presentation. The diagnostic tests have high specificity but a low sensitivity even in the presence of active tuberculosis (TB). A battery of tests may be required to arrive at the diagnosis (Tripathy and Tripathy, 1990; Jindal, 2006; Rozati et al., 2006). The fibrosis and scarring which result as a part of healing lead to the loss of function of the Fallopian tubes, and less commonly of ovaries and endometrium. It is therefore desirable to diagnose and treat GTB as early as possible during the subclinical stage to prevent or at least to minimize the damage to the genital organs. Unfortunately, the conventional tests for the diagnosis of TB during the sub-clinical stages have poor sensitivity and specificity. However, recently the detection of MTB DNA by TB-PCR has shown high sensitivity and specificity for the diagnosis of GTB (Baum et al., 2001; Roy et al., 2003; Bhanu et al., 2005; Rana et al., 2011). We have previously shown that the maximum likelihood estimates of sensitivity and specificity for the diagnosis of GTB with a positive TB-PCR in the endometrial samples were 0.59 and 0.92, respectively (Jindal et al., 2010). In a recent study, the authors have shown that 57% of infertile women in whom the presence of TB was suspected on clinical grounds had a positive endo-TB-PCR test, whereas only 9.5% had a positive test where no clinical ground for suspicion were present (Thangappah et al., 2011). Endometrial TB-PCR (endo-TB-PCR) positivity in the absence of symptoms, and without any demonstrable tubal or endometrial damage, raises the possibility of a false-positive TB-PCR test in the absence of any mycobacterial infection. Empirical treatment, especially in the high-prevalence countries is also fraught with the risks of resistance and other side effects of anti-tubercular chemotherapy. On the other hand, a positive endo-TB-PCR test may also imply the presence of sub-clinical, latent or past disease, which could be managed with anti-tubercular treatment (ATT). This is supported by the limited observations made for both genital and other forms of TB in some recent studies (Cheng et al., 2004; Kulshrestha et al., 2011; Thangappah et al., 2011). Short course chemotherapy is effectively used to treat symptomatic GTB (Jindal et al., 1990). However, for infertile women with GTB, assisted reproduction techniques (ARTs) are also required to achieve pregnancy (Soussis et al., 1998; Jindal, 2006; Singh et al., 2008). The present study was undertaken to examine the fertility of infertile women with positive endo-TB-PCR in the absence of demonstrable damage to the endometrium or the Fallopian tubes after the early institution of ATT.
Materials and Methods
The study was undertaken from the year 2006 to 2010 at an IVF center in northern India. Women from all couples seeking treatment for infertility between 2006 and 2008 were screened for inclusion in the study. All couples were investigated and managed according to the standard protocol followed at the center. Tubal and endometrial evaluation was done either by hysterosalpingography (HSG) or laparoscopy and hysteroscopy. Endometrial samples were obtained by endometrial aspiration or curettage, done as a stand-alone test or along with laparoscopy and/or hysteroscopy. One part of the biopsy of endometrial tissue was subjected to histopathologic examination and the second part was sent to the Laboratory for TB-PCR testing. Endometrial samples were obtained by gentle curettage of the endometrium and kept in sterile containers with normal saline to avoid contamination. Histopathologic examination of endometrial biopsies did not reveal any presence of acid fast bacilli, granuloma formation or other findings suggestive of TB.
PCR testing
Endo-PCR test was arranged with Reliance Life Sciences Pvt. Ltd. Mumbai, a national laboratory providing services all over India, accredited by the American College of Pathologists, with a proficiency test score for TB of 100% in 2007 and 2008. Stringent criteria were used at the laboratory to avoid contamination of results. The test was run in duplicate. Nested PCR against the most conserved region insertion sequence 6110 gene was done employing a Fastprepw sample preparation system (BioMedicals, Cambridge, UK) for mycolic acid cell wall lysis to extract DNA. The PCR assay gave a clear band of 123 base pairs (bp), indicating positivity of a sample. The assay used a cellular gene to rule out false negativity of the samples, with the cellular gene confirming no general DNA degradation in the sample and absence of PCR inhibitors in the sample. One positive and one negative clinical samples were used in every assay to validate the assay and confirm the results. A blank reagent containing no DNA was used to check contamination during PCR by the absence of any PCR fragment in the gel. The assay was also validated by direct sequencing of the PCR product indicating .95% homology with MTB, using NBLAST (www .ncbi.nlm.nih.gov/ blast). The quoted sensitivity of the test is almost 100% and specificity 96 – 99%, with a lower detection limit of 100 TB bacilli/ml (Nolte et al., 1993; Folgueira et al., 1996; Takahashi and Nakayama, 2006)
Patients
Of 3108 infertile couples who reported between 2006 and 2008, the study included 443 (14.2%) women of, 40 years of age who had no symptoms other than infertility, and without any evidence of endometrial or tubal damage on HSG or laparoscopy and hysteroscopy. The reasons for exclusion of the remaining 2665 (85.8%) cases were TB-PCR not done (1244); presence of a tubal factor (552); tubal evaluation not done (248); severe male factor (132); severe and moderate endometriosis (101); endometrial factor (76); previous history of ATT (168); age .40 or previous oophorectomy (91) and those who did not report for followup evaluation (53). Among 443 women who were included, 169 (38.15%) with PCR positive test constituted the study group (Group I) and 274 (61.85%) PCR negative, the control group (Group II). Follow-up assessment was continued for at least 2 years after recruitment in the study up to end of 2010.
Treatment
The study Group I, received standard short course daily ATT consisting of the intensive phase of 2 months of four drugs (isoniazid, H 300 mg; rifampicin, R 450 – 600 mg; ethambutol, E 800 – 1200 mg and pyrazinamide, Z 1200 – 1500 mg) followed by the maintenance phase of 4 months comprising the same doses of isoniazid and rifampicin (2HRZE, 4HR). The first-line treatment for non-tubal infertility consisted of ovulation induction along with IUIin PCR negative Group II and also in PCR positive Group I, after completion of ATT. IVF was undertaken for refractory infertility for 32 of Group I and 45 of Group II women; the remaining 150 women did not opt for IVF and wished to wait longer for various personal reasons. In brief, the IVF procedure consisted of employing stimulation protocols using GnRH analogs and urinary or recombinant gonadotrophins. Final maturation trigger was given when at least three lead follicles were .16 mm. Ovum pick up was done 36 h after hCG. All metaphase-2 oocytes were injected with sperm by intracytoplasmic sperm injection. Embryo transfer was done on second/third day and a maximum of three embryos were transferred. Pregnancy was defined as the presence of viable gestational sac on ultrasound examination at 3 – 4 weeks after embryo transfer.
Statistical methods
Details of all cases were recorded on a structured format and analyzed with the help of registered version of SPSS version 13. Group comparisons were made using x2 test (for categorical variables) or Student t-test (for scalar variables). Statistical significance was assessed at P , 0.05. Probability of spontaneous pregnancy (without any IUI or IVF) during the follow-up period was calculated by Kaplan –Meier method, and formal comparisons between different groups were performed using the log-rank test.
