Clinical Cohort Study on the Endocrinology and Vaginal/Endometrial Microbiome of the Luteal Phase in Assisted Reproduction

Introduction and rationale Progesterone is a steroid hormone with different functions. In the luteal phase of the menstrual cycle elevated progesterone levels maintain the endometrium. Amongst others it stimulates glandular secretion and adjusts pattern of secreted proteins of the endometrium to provide a supportive environment for of embryo implantation. A sudden decline in progesterone levels leads to endometrial shedding and thereby menstrual bleeding. Thus, elevated progesterone levels are vital to maintain a pregnancy after successful embryo implantation. Progesterone is produced by the Corpus luteum (CL), a relict of the Graaf follicle, after ovulation in the luteal phase of the menstrual cycle and in the first weeks of pregnancy. The lifespan of the CL is assumed to be several weeks.

Before luteolysis of the CL, the production of progesterone shifts from the CL to the placenta which ensures maintenance of the pregnancy. This process is termed luteoplacental shift. Adequate placental progesterone increase is vital for the maintenance of the pregnancy and low progesterone levels can indicate inadequate development of early pregnancies. However, laboratory measurement of progesterone by conventional ELISA techniques cannot distinguish between placental progesterone and progesterone produced by the CL.

This is one reason why still little is known about the exact time point of the luteoplacental shift. In 1972 a decline of progesterone and subsequent loss of pregnancy for n=12 patients after ovariectomy or luteectomy in the 8th week of gestation but not for operations taking place in the 9th week of gestation (n= 5 patients) was reported. This is in line with in-vitro measurements from 1985 in a placental organ culture which shows the capability for progesterone production between 6th and 8th week of gestation. In 1990 it was observed in women (n=17) with absent of ovaries and constant exogenous progesterone administration achieving pregnancy by an egg donation program a significant progesterone increase in the 9th gestational week. This is in contrast to a study in a similar setting from 1991 in n=9 women who reported onset of endogenous progesterone production around the 5th week of gestation. Additionally, even the existence of the luteoplacental shift itself was questioned because of a wide range of progesterone levels observed in women achieving successful pregnancies by assisted reproduction technique (ART).

Moreover, 17-OH progesterone (17-OHP) was suspected to be produced solely by the CL in early pregnancy. This is supported by a study who found 17-OHP blood levels and vascularity of the CL decreasing from 5th till 11th week of gestation suggesting the luteoplacental shift to take place.

In summary, little is known about the exact time point of the luteoplacental shift.

Secondly, to date the slope and magnitude of the placental progesterone increase and its relevance as indicator for the latter course of the pregnancy has not been properly studied.

In routine care patients undergoing transfer of cryopreserved embryos subsequently to an IVF/ICSI cycle use estradiol and progesterone supplementation to ensure anovulation during the menstrual cycle for optimal timing of embryo transfer. For long the vaginal application of micronized progesterone have been standard of care for this purpose. In March 2017 the LOTUS I trial showed non-inferiority for oral intake (3 x 10 mg) of dydrogesterone, a retroprogesterone, a same safety profile and a higher live birth rate of approximately +5% versus micronized vaginal progesterone for patients undergoing ART. These findings are supported by a Cochrane review comprising 94 randomized trials. Therefore, in routine care the standard regime for luteal support (LPS) for all ART patients was changed to oral intake of 30 mg dydrogesterone daily at the Department of Gynecological Endocrinology and Reproductive Medicine of the University of Luebeck.

Unlike micronized progesterone the chemical properties of dydrogesterone preclude detection in laboratory progesterone measurement apart from a small fraction of cross-reactivities. This circumstance allows an analysis of endogenous progesterone despite supplementation of dihydrogesteron at the same time.

In patients undergoing transfer of cryopreserved embryos utilization of a dydrogesterone regime for LPS provides the unique opportunity to study in detail the time point and magnitude of endogenous progesterone production (i.e. the luteal shift). The choice of protocol (supplementation with exogenous sex steroids or natural cycle) is taken, when the treatment is planned based on regularity of the cycle and patient preferences.

Recent studies have shown an influence of abnormal vaginal microbiota for the prediction of pregnancy and for preclinical pregnancy loss in IVF treatment. Therefore, this study aims to investigates possible crosslinks between endocrine profile, vaginal and endometrial microbiota and the establishment and maintenance of pregnancy.

Furthermore, possible differences in vaginal bleeding pattern between different groups of cryopreservation regimes have not been evaluated so far. This study aims to investigate whether vaginal bleeding patterns might be influenced the cryopreservation regime.