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Kisspeptin: A Potential Factor for Unexplained Infertility and Impaired Embryo Implantation

Background Kisspeptin (KP) is a neuropeptide that causes the release of the gonadotropin releasing hormone, which controls hypothalamo pituitary ovarian axis and exerts a number of peripheral effects on reproductive organs. The primary objective of this study was to compare baseline KP levels in females with different types of infertility and identify possible correlations with risk of failure to conceive, preclinical abortion and pregnancy after intracytoplasmic sperm injection (ICSI). Materials and Methods A longitudinal cohort study was carried out from August 2014 until May 2015 by recruiting 124 female patients undergoing ICSI, after obtaining ethical approval from the Australian Concept Infertility Medical Center. Cause of infertility due to male, female and unexplained factors was at a frequency of 32 (24%), 33 (31%) and 59 (45%) among the individuals respectively. KP levels were measured by ELISA assay before the initiation of the ICSI treatment protocol. Outcome of ICSI was categorized into three groups of non-pregnant with beta-human chorionic gonadotropin (β-hCG)<5-25 mIU/ml, preclinical abortion with β-hCG>25 mIU/ml and no cardiac activity, and clinical pregnancy declared upon confirmation of cardiac activity. Results based on cause of infertility and outcome groups were analyzed by one-way ANOVA. Results Females with unexplained infertility had significantly lower levels of KP when compared with those with male factor infertility (176.69 ± 5.03 vs. 397.6 ± 58.2, P=0.001). Clinical pregnancy was observed in 28 (23%) females of which 17 (71%) had a female cause of infertility. In the non-pregnant group of 66 (53%) females, common cause of infertility was unexplained 56(85%). A weak positive correlation of KP levels with fertilized oocytes and endometrial thickness was observed (P=0.04 and 0.01 respectively). Conclusion Deficiency of KP in females with unexplained infertility was associated with reduced chances of implantation after ICSI.


Introduction
Infertility has been recognized as a disease that requires timely diagnosis, recognition and treatment in terms of assisted reproduction. The preva--couples, male factors are diagnosed as the cause of infertility (2). Regarding females, structural causes of infertility include tubal damage due to uterine anomalies (3). The dysfunction of the hypothalamic pituitary ovarian axis (HPO) is also thought to be a key player resulting in infertility in many such situations. However, infertility in the presence of normal semen parameters, ovulatory concentration of serum progesterone in mid-luteal phase, tubal patency and a normal uterine cavity is often diagnosed as unexplained infertility (4). Assisted reproductive technology (ART) comprise in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with the latter shown to have a higher success rate (5). With both ARTs, higher failure is observed in couples with unexplained infertility as compared with other infertile subgroups, suggesting a need to explore other key factors that may play a role even in the absence of known abnormalities (6). Furthermore, studies linking unexplained infertility with higher rate of failure to implant after ART highlight the role of sisting the invasion of the uterine wall (7). In general, the subfertile population is at a greater risk of preclinical abortion. Likewise, its frequency pre-ART. Both decreased receptivity of the uterus and chromosomal abnormality in the fetuses are thought to trigger this response, however, other factors that may lead to loss of pregnancy between the time of implantation and onset of menses still remains unknown (8).
Recently, the role of serum kisspeptin (KP) in regulating HPO axis and maturity of oocytes has been aptly investigated (9). KP, an RF-amide peptide coded by the KISS1 gene, was initially believed to be a tumor suppressor gene. The KP receptor, previously known as GPR54, plays an important role in maintaining fertility in both huof gonadotrophin releasing hormone (GnRH) neurons express the GPR54 receptors (11). Neurons located in the hypothalamus secrete KP, thus triggering KP receptors (GPR54) on the GnRH neurons and leading to the secretion of GnRH (9). Besides this, KP is triggered at the onset of puberty and relays information about energy stores of the body to the central nervous system (CNS) by modulating negative and positive feedback of gonadal steroids. Aside from its wide distribution in the brain, KP receptors are highly expressed in the pancreas, placenta, uterus, small intestine, kidney, lung, liver and heart (12). Studies have also suggested other roles for Kisspeptin such as the emergence of KP as a key regulator of the central mammalian reproductive axis along with its role in placentation and pregnancy. This has lead to the exploration of its probable therapeutic role in treating certain forms of infertility (9, 13). We therefore aimed to study the levels of baseline serum KP in infertile females with various types of infertility and examine whether it correlates with the risk of failure to conceive, preclinical abortion and pregnancy after assisted reproduction by ICSI.

Materials and Methods
A longitudinal cohort study was carried out from was obtained from the Australian Concept Infertility Medical Center. One hundred and twentyfour female patients were recruited after receiving their written informed consent to participate in 2 , were segregated into three groups on the basis of their infertility and all were recommended for ICSI. Cause of infertility due to male, female and unexplained factors was at a frequency of 32 (26%), 33 consisted of those with a male factor infertility (e.g varicocele, prior surgeries and semen abnormalities). The second group comprised females with comprised 59 patients with unexplained infertility. Females with metabolic disorder (polycystic ovaries) and endocrine disorders such as thyroid dysfunction and abnormal prolactin levels were excluded from the study. Couples diagnosed with both male and female infertility factors were excluded from the study.
Serum samples were collected on the second day of the menstrual cycle before commencement of down-regulation of ovaries. KP was measured using the KISS-1-ELISA Kit (Shanghai, China). The analytical sensitivity of the KP respectively.
Down-regulation of ovaries by 1 mg of subcuta-21 of previous menstrual cycle was followed by controlled ovarian stimulation with Gonal-f (Merck was assessed by transvaginal scan (TVS) and cycles were cancelled when follicles failed to develop in response to gonadotropin stimulation. Endometrial thickness was gauged on the day of ovulation induction in the mid sagittal plane by two-dimensional ultrasound with a 7.5-MHz vaginal probe (Hitachi EUB 525, Hitachi, Japan). Oocytes were retrieved hours after injection of human chorionic gonadotroth , 15 th or 16 th day of stimulation. Semen samples were retrieved by masturbation and sperms were then immobilized by 7% polyvinyl pyrrolidone after which microinjection was performed (Leica DMIRB, Leica Microsystems, Wetzlar, Germany). Finally, the microinjected oocytes were incubated for 16-18 hours at 2 and 5% O 2 .
concentrations on the 14 th day after egg collection and on the basis of sonographic evidence of an were declared as non-pregnant, pre-clinical abortion was labelled on a beta hCG>25 m IU/ml but with no cardiac activity on ultrasound while clini-els of beta hCG along with intrauterine gestational sac and cardiac activity on TVS. The implantation rate (IR) was calculated as the number of pregnancies per embryo transferred (14), clinical pregnancy (CP) was established by the presence of an number of patients at the start of cycle (15).
Data were analyzed by SPSS version 21 (IBM statistics, Chicago, IL) and was expressed as appropriate. Analyses were undertaken for the whole group and independently in sub-groups according to the infertility factor. Variation in KP levels in females with different types of infertility and different outcomes after ICSI was compared by Analysis of variance (ANOVA) and Tukey's was used to test correlation between KP levels Table 1 summarises the characteristics of the study population with respect to type of infertil-duced levels in female and unexplained infertility factor sub-groups when compared with those with respectively).

Implantation rate
Blood collected on the second day of cycle before initiation of stimulation. Data expressed as mean ± SD, except kisspeptin levels expressed as mean ± SEM.

Fig.1:
Although variation in KP levels between nonpregnant and pre-clinical abortion groups was comes of ICSI treatment based on type of infertility are presented in Table 2. Highest number of clinical pregnancies was achieved in the group of infertile females with diagnosed cause of infertility (51%), however, only 5% of patients with unexplained infertility got pregnant. A weak but tween KP levels and number of fertilized oocytes -

Discussion
With the advancement of ART, couples have been able to conceive, nevertheless, the outcome is associated with a limited success rate (14). The treatment procedures are equally offered to all females suffering from known and unknown factors that disrupt effective functioning of reproductive organs. We observed the lowest levels of KP in females who had unexplained infertility which is consistent with the hypothesis that mutation in the in Exon 1 of KISS1 may disrupt HPO axis, reduce KP levels and likely to be one of the reasons of unexplained infertility (16). Cytokines play an active role in folliculogenesis, ovulation, fertilization and implantation (17). KP is known to work as a leading to implantation (18,19). Many studies have linked unexplained infertility with low implantation rates while highlighting the role of interleukins sion of the uterine wall (7). In our study, reduced KP levels in females with unexplained infertility was associated with inadequate egg maturation, reduced fertilization, thinner endometrial lining and failure of implantation of the blastocyst. This is probably the basis by which injection of a single dose of KP-54 triggers egg maturation, fertilization and implantation of blastocyst in infertility treat-  Fayazi et al. (22) discovered the presence of a KP/KISS1R signaling pathway in the uterus of 4-day pregnant mouse with a slight expression of KP signaling in the uterus of non-pregnant mice. KP expression thus has a maintenance basal level in peripheral reproductive organs, which is up-regulated at the initiation of pregnancy. A low level of KP in non-pregnant females in our study emphasizes its role in placentation and implantation. This observation is in accordance with the researchers previous work with the difference that KP was estimated after the suppression of HPO axis (23). Preclinical abortion after ICSI is subject to a number of factors including the etiology of infertility (8). The preclinical abortion was observed in 24% of the study population, which mainly comprised and tubal blockade. These results are contradictory to those in studies that observed similar pregnancy loss with different types of infertility (24). We nonetheless observed that women with preclinical abortions had a higher KP level than patients who failed to conceive. This may be explained by the existence of a circadian KP expression in a fullterm human placenta of healthy women that regulates trophoblastic invasion and probably pregnancy maintenance (25).
During the menstrual cycle, secretory changes in the endometrium account for coordinated signal exchange between hormonally primed endometrium and functional embryo for the implantation of embryo. This is made possible by the interplay of hormones and cytokines, and studies have demonstrated that increased endometrial thickness is associated with higher pregnancy rates (26). A cut-off value of 8mm thickness was considered to be optimal for embryo implantation after ICSI in the female population of Pakistan (17). We observed that a thicker endometrial lining was associated with a higher implantation rate when compared with females who could not conceive at all. Moreover, thickness of the endometrium was at its lowest in patients with unexplained infertility, who also had minimum levels of serum KP.
The prevalence of infertile couples with unexsupports association of reduced KP in unexplained infertile females with reduced fertilization of oocytes, implantation of blastocyst and hence con-ception. Improving the success rate after IVF and ICSI for couples with unexplained infertility is thus what further work should be aimed at. This study is uni-centric, has a small sample size and included infertile couples with both male and female factors, which all may bias the observed correlation between KP levels and unexplained infertility. In addition, we did not stratify the male infertility factor based on sperm parameters including count, motility and morphology. This may be a confounding factor for low implantation rate in females with diagnosed male infertility factor.

Conclusion
Low level of KP in females with unexplained infertility before the initiation of the ICSI protocol was seen. One of the factors causing unexplained infertility may thus be low KP levels. The level of KP has an impact on fertilization of oocytes, preparation of endometrial beds for implantation of embryo and hence successful pregnancy after ICSI. This observation urges the need of further and explain the role of KP for preservation of conception and continuation of pregnancy in females with unexplained infertility.